Detection of truncation mutations by mass spectrometry

ABSTRACT

This invention relates to the detection and analysis by mass spec of nascent proteins, and in particular truncated proteins, translated within cellular or cell-free translation systems. N-terminal and C-terminal epitopes introduced into these nascent proteins permit rapid and efficient isolation, as well as a mass difference.

FIELD OF THE INVENTION

This invention relates to assays and markers that facilitate thedetection and analysis of nascent proteins translated within cellular orcell-free translation systems. Nascent proteins (and in particular,truncated proteins) containing these markers can be rapidly andefficiently detected and analyzed by mass spectrometry.

BACKGROUND OF THE INVENTION

Detection of mutations is an increasingly important area in clinicaldiagnosis, including but not limited to the diagnosis of cancer and/orindividuals disposed to cancer. The protein truncation test (PTT) is atechnique for the detection of nonsense and frameshift mutations whichlead to the generation of truncated protein products. Genes associatedwith Duchenne muscular dystrophy, adenomatous polyposis coli, human mutLhomologue and human nutS homologue (both involved in colon cancer), andBRAC1 (involved in familial breast cancer) can now be screened formutations in this manner, along with others.

Typically, the PTT technique involves the incorporation of a T7 promotersite, ribosome binding site, and an artificial methionine start siteinto a PCR product covering the region of the gene to be investigated.The PCR product is then transcribed and translated using a cell-freetranslation system, such as an in vitro rabbit reticulocyte lysate,wheat germ lysate or E. coli lysate system, to generate a proteincorresponding to the region of the gene amplified. The presence of astop codon in the sequence, generated by a nonsense mutation or aframeshift, will result in the premature termination of proteintranslation, producing a truncated protein that can be detected bystandard gel electrophoresis (e.g. SDS-PAGE) analysis combined withradioactive detection.

There are drawbacks to the technique as currently practiced. One of themost important problems involves the identification of the product ofinterest. This is made difficult because of nonspecific radiolabeledproducts. Attempts to address these problems have been made. Oneapproach is to introduce an affinity tag after the start site and beforethe region encoding the gene of interest. See Rowan and Bodmer,“Introduction of a myc Reporter Taq to Improve the Quality of MutationDetection Using the Protein Truncation Test,” Human Mutation 9:172(1997). However, such approaches still have the disadvantage that theyrely on electrophoresis.

SUMMARY OF THE INVENTION

The present invention contemplates an assay wherein two or three markers(preferably N-terminal and C-terminal epitopes) are introduced into thenascent protein and the resulting wild-type and mutant proteins aredetected by mass spectrometry. In a preferred embodiment of theinvention, the novel compositions and methods are directed to thedetection of frameshift or chain terminating mutations associated withdisease. It is not intended that the present invention be limited todetecting mutations to only one particular disease. A variety ofdiseases are linked to such mutations (see Table 1) and are, therefore,contemplated.

In order to detect such mutations, a nascent protein (typically aportion of a gene product, wherein the portion is between 5 and 200amino acids in length, and more commonly between 5 and 100 amino acidsin length, and more preferably between 5 and around 60 amino acids inlength—so that one can work in the size range that corresponds tooptimal sensitivity on most mass spectrometry equipment) is (in oneembodiment) first synthesized in a cell-free or cellular translationsystem from message RNA or DNA coding for the protein which may containa possible mutation. The nascent protein is then separated from thecell-free or cellular translation system using the N-terminal epitope(located at or close to the N-terminal end of the protein). Theresulting isolated material (which may contain both wild-type andtruncated peptides) is then analyzed by mass spectrometry. Detection ofa peak in the mass spectrum with a mass correlating with a peptidehaving the marker/epitope located at or close to the C-terminal of theprotein (C-terminal epitope) indicate the wild-type peptide. Detectionof a peak in the mass spectrum with a mass correlating with a peptidelacking a C-terminal marker indicates a truncating mutation. To enhancesensitivity, the C-terminal epitope in some embodiments can be used(prior to mass spec) to deplete wild-type sequences (i.e. enrich fortruncated proteins) by interacting with a ligand (e.g. an antibody)directed to the C-terminal epitope (e.g. affinity chromatography).Alternative methods of depleting wild type sequence are alsocontemplated involving the using of an affinity tag incorporated by amisaminoacylated tRNA. In one embodiment, a biotin tag is incorporatedin a sequence at or near the C-terminal end. This tag can be used inconjunction with streptavidin coated media to deplete a wild-typesequence.

In most cases, it is expected that the wild-type polypeptides will bepresent in a greater amount that the truncated polypeptides.Nonetheless, the present invention contemplates methods where thetruncated polypeptide is readily detected by mass spectrometry. In oneembodiment, the present invention contemplates a method, comprising:providing a preparation comprising wild type polypeptides and truncatedpolypeptides (preferably made in an in vitro translation reaction) in aratio of at least 10:1, wherein said truncated polypeptides are due to agenetic mutation and are between 10 and 100 amino acids in length (butmore typically between 20 and 80 amino acids in length, and moreconveniently between 30 and 60 amino acids in length); and determiningthe molecular mass of said truncated polypeptides by mass spectrometry.In some embodiments, the said wild type polypeptides and said truncatedpolypeptides are in a ratio of at least 50:1. In still otherembodiments, said wild type polypeptides and said truncated polypeptidesare in a ratio of at least 100:1. In a preferred embodiment, the methodfurther comprising the step of removing at least a portion of said wildtype polypeptides from said preparation prior to step (b). In aparticularly preferred embodiment, said wild type polypeptides comprisesa C-terminal epitope and said removing is achieved by exposing saidpreparation to a ligand with affinity for said C-terminal epitope. It ispreferred that at least a portion of each of said wild-type polypeptidesis identical to a portion of a disease-related gene product (e.g. APCgene product).

The creation of a stop codon from a frameshift mutation is random. Wherea stop codon is created, there is a significant difference in massbetween the proteins containing both the C-terminal marker andN-terminal marker (i.e. wild-type proteins) and the truncated proteinscontaining only the N-terminal marker. On the other hand, it is possiblethat a frameshift mutation near the C-terminus will not result in stopcodon. In a preferred embodiment, to ensure that full advantage is takenof this mass difference, a sequence (discussed below) is introducedadjacent the C-terminal epitope which will generate a stop codon ifthere is a frameshift. Such an approach does not rely on the randomformation of stop codons.

In a preferred embodiment, mass spectrometry provides information aboutthe fraction of nascent proteins containing frameshift or chainterminating mutations in the gene sequence coding for the nascentprotein. The amount of wild-type sequence (i.e. protein containing theC-terminal epitope) reflects the fraction of protein which did notcontain chain terminating or out-of-frame mutations.

Separating the protein(s) from the translation mixture (prior to massspectrometry) using an affinity marker located at or close to theN-terminal end of the protein eliminates the occurrence of false startswhich can occur when the protein is initiated during translation from aninternal AUG in the coding region of the message. A false start can leadto erroneous results since it can occur after the chain terminating orout-of-frame mutation. This is especially true if the internal AUG isin-frame with the message. In this case, the peptide C-terminal markerwill still be present even if message contains a mutation.

It is not intended that the present invention be limited to the sourceof nucleic acid. A variety of sources are contemplated (e.g. tissuesamples from a biopsy), including but not limited to nucleic acid fromblood and stool samples. Humans of all ages can be so tested in arelatively non-invasive manner. Both the existence of disease and thepredisposition to disease can be tested. For example, in one embodiment,the present invention contemplates both pre-natal (e.g. amniotic fluid)and post-natal testing to determine predisposition to disease.

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides methods forthe labeling, isolation, detection, quantitation, and analysis ofnascent proteins produced in a cell-free or cellular translation systemwithout the use of gels or radioactive labels.

It is not intended that the present invention be limited to a particulartranslation system. In one embodiment, a cell-free translation system isselected from the group consisting of Escherichia coli lysates, wheatgerm extracts, insect cell lysates, rabbit reticulocyte lysates, frogoocyte lysates, dog pancreatic lysates, human cell lysates, mixtures ofpurified or semi-purified translation factors and combinations thereof.In a preferred embodiment, the system is a cell-free translation systemthat has been reconstituted with purified components (e.g. initiationfactors, elongation factors, termination factors, aminoacyl-tRNAsynthetase, methionyl-tRNA transformylase). See Y. Shimizu et al.,“Cell-free translation reconstituted with purified components,” NatureBiotechnology 19:751 (2001). See also U.S. patent application Ser. No.983,067, filed Oct. 23, 2001, hereby incorporated by reference.

While it is not intended that the present invention be limited to theparticular reaction conditions employed, some parameters need to be keptin mind. Typically the cell-free translation system is incubated at atemperature of between about 25° C. to about 45° C. (and preferably 37°C.). Importantly, it has been found that all commercially availabletranslation systems (including the reconstituted systems advertised asprotease-free) have significant protease activity. Certain proteaseinhibitors (discussed below) can be used to reduce this proteaseactivity—without significantly interfering with translation. Steps canbe taken to directly remove proteases (e.g. by immunoprecipitation withspecific antibodies or substrates). In addition, replacement of theribosome component with more highly purified ribosomes can reduceprotease activity. However, even with protease inhibitors and morepurified components, exposure of the nascent protein to the translationmixture for extended periods of time (e.g. an hour) is generally to beavoided.

To ensure that proteases are not complicating the analysis (e.g. causingfalse negatives by proteolyzing the truncated peptides or causing falsepositives by proteolyzing the wild-type peptides), the present inventioncontemplates the use of a control or reference peptide that is “proteasesensitive,” i.e. sensitive to the protease activity of the particulartranslation system (the protease activity of various systems isdescribed herein) such that partial protease digestion, e.g. at least20% (and more typically between 40-60%) of the peptides in thepopulation have had one or more amino acids removed, can be observedafter 10 minutes of exposure (or less) to the translation mixture at 37°C. Quantitation can be approximated simply by comparing peak heights inthe mass spectrum, with the understanding that factors influencing peakheight will be similar (but not identical) for the digested andundigested peptides. Quantitation can be better approximated by using anundigested control (i.e. a peptide that has not been exposed to thetranslation mixture); however, adding the control to the sample cancomplicate the analysis unless steps are taken to create a control thathas a mass that is distinct from undigested peptide (which has beenexposed to the translation mixture but was not proteolyzed). In oneembodiment, the control is an isotope labeled version of the proteasesensitive peptide. This permits the use of compounds that aresubstantially chemically identical, but isotopically distinguishable. Amethod for the production of molecules comprising deuterium atoms isgiven in U.S. patent application Ser. No. 2002/0119490 A1 and referencestherein, all of which are incorporated herein by reference. For example,one or more hydrogens in the peptide can be substituted with deuteriumto generate isotopically heavy reagents. Isotopically labeled aminoacids are commercially available (Cambridge Isotope Laboratories,Andover, Mass.) and can simply be used in peptide synthesis.

While a variety of peptide designs are possible, the present inventioncontemplates an embodiment wherein the protease-sensitive peptidecomprises an N-terminal epitope (for convenient capture and purificationfrom the mixture), a region of positively charged amino acids such asarginine, lysine or histidine (to improve flight in the mass spec), anda C-terminal region comprising hydrophobic amino acids (e.g.phenylalanine) for protease digestion. Optionally, the N-terminus canhave other amino acids (e.g. methionine) or protecting groups (FMOC,etc.). It is not intended that the present invention be limited by theparticular epitope; known epitopes (or variants thereof) can be employed(whether containing positively charged amino acids or not). The regionof positively charged amino acids can be a) a single amino acid (e.g.one arginine), or b) a plurality of amino acids. Where it is aplurality, it may be comprised of a mixture of different amino acids or(preferably) can be a string (e.g. between 2 and 20 amino acids,preferably between 2 and 9 amino acids, and more preferably between 5and 7 amino acids) of a single amino acid (e.g. arginine). Examples ofprotease-sensitive peptides where arginine is used (between one and nineamino acids) in the region of positively charged amino acids—along witha variety of epitopes—are shown in Table 2 (SEQ ID NOS: 1-109). Thepresent invention contemplates these peptides as compositions of matterand as useful in various assays described herein (including but notlimited to a mass spec-based protease detection assay which can be used,among other things, to quality control commercially availabletranslation systems).

While the protease-sensitive peptide can be made synthetically, it canalso be made during the translation process. Therefore, in oneembodiment, the present invention contemplates nucleic acid coding forthe protease-sensitive peptide as well as a method wherein said nucleicacid is used as a template for translation. Nucleic acid sequences (SEQID NOS: 110-119) for a number of epitopes (SEQ ID NOS: 120-129) areprovided in Table 3. An experimental example is described herein whereinthe protease-sensitive peptide is made during in vitro translation.

For high throughput, most of the steps can be readily automated. While abatch approach can be readily utilized, the present invention alsocontemplates both continuous flow systems or dialysis systems.

In a preferred embodiment, a transcription/translation system usedwherein nucleic acid (typically DNA, but RNA if desired) coding for theprotein which may contain a possible mutation is added to thetranslation system. The system is incubated to synthesize the nascentproteins. The nascent protein is then separated from the translationsystem using an affinity marker. In one embodiment the affinity markeris located at or close to (e.g. within ten amino acids of) theN-terminal end of the protein, while in another embodiment, the affinitymarker can be distributed throughout the sequence of the protein(whether randomly or at defined intervals).

It is not intended that the present invention be limited by the natureof the N- and C-terminal epitopes, or the type of affinity markerutilized. A variety of markers are contemplated. Table 3 provides anumber of commercially available epitopes (and additional epitopes aredescribed in the examples). In one embodiment, the affinity markercomprises an epitope recognized by an antibody or other bindingmolecule. In another embodiment, the affinity marker is biotin and isdistributed randomly on lysine residues. In one embodiment, theN-terminal marker comprises a fluorescent marker (e.g. a BODIPY marker),while the C-terminal marker comprises a metal binding region (e.g. Histag).

The present invention contemplates a variety of methods wherein two orthree markers (e.g. the N- and C-terminal markers and the affinitymarkers) are introduced into a nascent protein. In both the two markerand three marker embodiments, the present invention contemplates thatone or more of the markers (and in the preferred embodiment, all of themarkers/epitopes) will be introduced into the nucleic acid template byprimer extension or PCR (and thus, introduction via charged tRNAs isunnecessary). In one embodiment, the present invention contemplates aprimer comprising (on or near the 5′-end) a promoter, a ribosome bindingsite (“RBS”), and a start codon (e.g. ATG), sequence encoding anepitope, along with a region of complementarity to the template (e.g.sufficiently complementary to hybridize to a portion of adisease-related gene or, in preferred embodiments, completelycomplementary to a portion of a disease-related gene). In anotherembodiment, the present invention contemplates a primer comprising (onor near the 5′-end) a promoter, a ribosome binding site (“RBS”), a startcodon (e.g. ATG), a region encoding a second epitope, and a region ofcomplementarity to the template. It is not intended that the presentinvention be limited by the length of the region of complementarity;preferably, the region is greater than 8 bases in length, morepreferably greater than 15 bases in length, and still more preferablygreater than 20 bases in length (but commonly less than 40 bases inlength, and more typically less than 30 bases in length).

It is also not intended that the present invention be limited by theribosome binding site. In one embodiment, the present inventioncontemplates primers comprising the Kozak sequence, a string ofnon-random nucleotides (consensus sequence 5′-GCCA/GCCATGG-3′) (SEQ IDNO:130) which are present before the translation initiating first ATG inmajority of the mRNAs which are transcribed and translated in eukaryoticcells. See M. Kozak, Cell 44:283-292 (1986). In another embodiment, thepresent invention contemplates a primer comprising the prokaryotic mRNAribosome binding site, which usually contains part or all of apolypurine domain UAAGGAGGU (SEQ ID NO:131) known as the Shine-Dalgamo(SD) sequence found just 5′ to the translation initiation codon: mRNA5′-UAAGGAGGU-N₅₋₁₀-AUG. (SEQ ID NO:132)

For PCR, two primers are used. In one embodiment, the present inventioncontemplates as the forward primer a primer comprising (on or near the5′-end) a promoter, a ribosome binding site (“RBS”), a start codon (e.g.ATG), a region encoding an affinity marker, and a region ofcomplementarity to the template. The present invention contemplates thatthe reverse primer, in one embodiment, comprises (at or near the 5′-end)one or more stop codons and a region encoding a C-terminus marker (suchas a HIS-tag) and (optionally) a region which will generate a stop codonif there is a frameshift. This latter region can be designed in a numberof ways. However, to efficiently generate stop codons for every type offrameshift, the following sequence is useful: ATA-AAT-AAA (SEQ IDNO:133). Where there are no frameshifts, this sequence codes forIle-Asn-Lys (SEQ ID NO:134). Where there are frameshifts, the sequencewill generate a stop codon. Since the sequence is preferred as part ofthe reverse primer, the sequence is used in the following orientation:5′-TTT-ATT-TAT-3′ (SEQ ID NO:135).

The present invention also contemplates embodiments where the affinitymarker is introduced through a misaminoacylated tRNA. In one embodimentthe misaminoacylated tRNA only recognizes a codon which codes for aparticular amino acid such as a codon for lysine. In this case, theaffinity marker is incorporated randomly throughout the proteinsequence. In another embodiment, more than one misaminoacylated tRNA isutilized. In this case, the affinity marker may be randomly distributedthroughout the protein sequence at more than a single amino acid such aslysine or tyrosine. In another embodiment the misaminoacylated tRNA is asuppressor tRNA and incorporates the affinity marker at a specificposition in the protein sequence.

Another aspect of the present invention contemplates an oligonucleotide,comprising a 5′ portion, a middle portion contiguous with said 5′portion, and a 3′ portion contiguous with said middle portion, whereini) said 5′ portion comprises a sequence corresponding to a promoter, ii)said middle portion comprises a sequence corresponding to a ribosomebinding site, a start codon, and a sequence coding for an epitope marker(or variant thereof that can be recognized by an antibody), and iii)said 3′ portion comprises a sequence complementary to a portion of theAPC gene (or another gene whose truncated products are associated withdisease, i.e. a “disease related gene”). In one embodiment, saidoligonucleotide is less than two hundred bases in length. In a preferredembodiment, said oligonucleotide is less than one hundred bases inlength, and most preferably less than 70 bases in length (e.g. between40 and 60 bases in length). In one embodiment, said 5′ portion isbetween ten and forty bases in length (preferably between eight andsixty bases in length, and more preferably between fifteen and thirtybases in length). In one embodiment, said middle portion is between tenand one hundred bases in length (preferably between eight and sixtybases in length, and more preferably between fifteen and thirty bases inlength). In one embodiment, said 3′ portion is between ten and fortybases in length (and more preferably between fifteen and thirty bases inlength). In one embodiment, said sequence complementary to the portionof the APC gene is greater than 15 bases in length. In anotherembodiment, said sequence complementary to the portion of the APC geneis greater than 20 bases in length.

Another aspect of the present invention contemplates a kit, comprising:a) a first oligonucleotide comprising a 5′ portion, a middle portioncontiguous with said 5′ portion, and a 3′ portion contiguous with saidmiddle portion, wherein i) said 5′ portion comprises a sequencecorresponding to a promoter, ii) said middle portion comprises asequence corresponding to a ribosome binding site, a start codon, and asequence coding for a first epitope marker, and iii) said 3′ portioncomprises a sequence complementary to a first portion of the APC gene(or other disease related gene); b) a second oligonucleotide comprisinga 5′ portion, a middle portion contiguous with said 5′ portion, and a 3′portion contiguous with said middle portion, wherein i) said 5′ portioncomprises at least one stop codon, ii) said middle portion comprises asequence encoding for a second epitope marker, and iii) said 3′ portioncomprises a sequence complementary to a second portion of the APC gene(or other disease related gene). Optionally, the kit comprises aprotease-sensitive peptide (discussed above) to be used as a control formass spec. In one embodiment, said kit further comprises a polymerase.In another embodiment, said kit further comprises a misaminoacylatedtRNA. In another embodiment, said kit further comprises antibodiesdirected against said epitopes.

Another aspect of the present invention contemplates a method ofintroducing coding sequence for one or more epitope markers into nucleicacid, comprising: a) providing: a first oligonucleotide primercomprising a 5′ portion, a middle portion contiguous with said 5′portion, and a 3′ portion contiguous with said middle portion,wherein 1) said 5′ portion comprises a sequence corresponding to apromoter, 2) said middle portion comprises a sequence corresponding to aribosome binding site, a start codon, and a sequence coding for a firstepitope marker, and 3) said 3′ portion comprises a sequencecomplementary to a first portion of the APC gene (or other diseaserelated gene); ii) a second oligonucleotide primer comprising a 5′portion, a middle portion contiguous with said 5′ portion, and a 3′portion contiguous with said middle portion, wherein 1) said 5′ portioncomprises at least one stop codon, 2) said middle portion comprises asequence encoding for a second epitope marker, and 3) said 3′ portioncomprises a sequence complementary to a second portion of the APC gene(or other disease related gene); iii) a polymerase; and iv) templatenucleic acid comprising a region of the APC gene (or other diseaserelated gene), said region comprising at least said first portion of theAPC gene; and b) mixing said template nucleic acid with said firstprimer, second primer and said polymerase under conditions such thatamplified template is produced, said amplified template comprising saidsequence coding for an epitope marker. In one embodiment, said first andsaid second oligonucleotide are each less than one hundred bases inlength. In one embodiment, said sequence complementary to a portion ofthe APC gene of said first and said second oligonucleotide is 10 basesor greater, but preferably greater than 15 bases in length. In anotherembodiment, said sequence complementary to a portion of the APC gene ofsaid first and said second oligonucleotide is greater than 20 bases inlength.

While not intending to limit the present invention, it is understood byone skilled in the art, that “a region of the APC gene” is larger than“a portion of the APC gene” (just as “regions” of any other geneassociated with disease are larger than “portions” of the same). Forexample, a region of the APC gene may comprise, but is not limited to,the region coding for amino acids 1098-1696 (i.e., segment 3). Othersegments (such as segment 23) are also contemplated.

Another aspect of the present invention contemplates a method,comprising: a) providing: i) the amplified template (described above);i) a translation system; b) introducing said amplified template intosaid translation system so as to create nascent protein (or portionthereof) comprising an N-terminal epitope; c) isolating said nascentprotein; and d) detecting said protein (or portion thereof) by massspectrometry.

In one embodiment, the isolating of the nascent protein comprisesimmobilizing said nascent protein by contacting said nascent proteinwith a ligand (e.g. antibody) which binds the N-terminal epitope.Typically, said ligand is attached to a solid support. Where theN-terminal epitope is biotin (for example), said ligand is selected fromthe group consisting of avidin and strepavidin, and variants, mutantsand derivatives thereof.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part, will be obviousfrom this description, or may be learned from the practice of theinvention.

Definitions

To facilitate understanding of the invention, a number of terms aredefined below.

“Proteins” are composed of “amino acids” arranged into linear polymersor polypeptides. In living systems, proteins comprise over twenty commonamino acids. These twenty or so amino acids are generally termed the“native” amino acids. At the center of every amino acid is the alphacarbon atom which forms four bonds or attachments with other molecules(FIG. 1). One bond is a covalent linkage to an amino group (NH₂) andanother to a carboxyl group (COOH) which both participate in polypeptideformation. A third bond is nearly always linked to a hydrogen atom andthe fourth to a side chain which imparts variability to the amino acidstructure. For example, alanine is formed when the side chain is amethyl group (—CH₃) and a valine is formed when the side chain is anisopropyl group (—CH(CH₃)₂). It is also possible to chemicallysynthesize amino acids containing different side-chains, however, thecellular protein synthesis system, with rare exceptions, utilizes nativeamino acids. Other amino acids and structurally similar chemicalcompounds are termed non-native and are generally not found in mostorganisms.

A central feature of all living systems is the ability to produceproteins from amino acids. Basically, protein is formed by the linkageof multiple amino acids via peptide bonds such as the pentapeptidedepicted in FIG. 1B. Key molecules involved in this process aremessenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules andribosomes (rRNA-protein complexes). Protein translation normally occursin living cells and in some cases can also be performed outside the cellin systems referred to as cell-free translation systems. In eithersystem, the basic process of protein synthesis is identical. Theextra-cellular or cell-free translation system comprises an extractprepared from the intracellular contents of cells. These preparationscontain those molecules which support protein translation and dependingon the method of preparation, post-translational events such asglycosylation and cleavages as well. Typical cells from which cell-freeextracts or in vitro extracts are made are Escherichia coli cells, wheatgerm cells, rabbit reticulocytes, insect cells and frog oocytes.

Both in vivo and in vitro syntheses involve the reading of a sequence ofbases on a mRNA molecule. The mRNA contains instructions for translationin the form of triplet codons. The genetic code specifies which aminoacid is encoded by each triplet codon. For each codon which specifies anamino acid, there normally exists a cognate tRNA molecule whichfunctions to transfer the correct amino acid onto the nascentpolypeptide chain. The amino acid tyrosine (Tyr) is coded by thesequence of bases UAU and UAC, while cysteine (Cys) is coded by UGU andUGC. Variability associated with the third base of the codon is commonand is called wobble.

Translation begins with the binding of the ribosome to mRNA (FIG. 2). Anumber of protein factors associate with the ribosome during differentphases of translation including initiation factors, elongation factorsand termination factors. Formation of the initiation complex is thefirst step of translation. Initiation factors contribute to theinitiation complex along with the mRNA and initiator tRNA (fmet and met)which recognizes the base sequence UAG. Elongation proceeds with chargedtRNAs binding to ribosomes, translocation and release of the amino acidcargo into the peptide chain. Elongation factors assist with the bindingof tRNAs and in elongation of the polypeptide chain with the help ofenzymes like peptidyl transferase. Termination factors recognize a stopsignal, such as the base sequence UGA, in the message terminatingpolypeptide synthesis and releasing the polypeptide chain and the mRNAfrom the ribosome.

The structure of tRNA is often shown as a cloverleaf representation(FIG. 3A). Structural elements of a typical tRNA include an acceptorstem, a D-loop, an anticodon loop, a variable loop and a T1C loop.Aminoacylation or charging of tRNA results in linking the carboxylterminal of an amino acid to the 2′-(or 3′-) hydroxyl group of aterminal adenosine base via an ester linkage. This process can beaccomplished either using enzymatic or chemical methods. Normally aparticular tRNA is charged by only one specific native amino acid. Thisselective charging, termed here enzymatic aminoacylation, isaccomplished by aminoacyl tRNA synthetases. A tRNA which selectivelyincorporates a tyrosine residue into the nascent polypeptide chain byrecognizing the tyrosine UAC codon will be charged by tyrosine with atyrosine-aminoacyl tRNA synthetase, while a tRNA designed to read theUGU codon will be charged by a cysteine-aminoacyl tRNA synthetase. ThesetRNA synthetases have evolved to be extremely accurate in charging atRNA with the correct amino acid to maintain the fidelity of thetranslation process. Except in special cases where the non-native aminoacid is very similar structurally to the native amino acid, it isnecessary to use means other than enzymatic aminoacylation to charge atRNA.

The term “portion” refers to something is “less than the whole” and thusmay refer to a relatively small part of a protein or an oligonucleotide.Specifically, a portion of a protein typically is in the range ofbetween 5-200 contiguous amino acids (assuming that the protein has morethan 200 amino acids) while a portion of a nucleic acid refers to arange of between 15-600 contiguous bases (again, assuming the gene hasmore than 600 contiguous bases). Smaller portions (e.g. less than 5amino acids) can be used but are not practical if one is attempted todetect mutations over a large area by examining a plurality of “testsequences”. For example, to cover segment 3 of the APC gene,amplification of ten (usually contiguous) test sequences (whetheroverlapping or non-overlapping) of this region are performed. Since massspec is used for analysis, the size of the test sequence is dictated inpart by the reliable mass range of the equipment (factoring in the sizeof the N- and C-terminal epitopes). If overlapping test sequences areemployed, the primers can be designed to hybridize inside the testsequence. If non-overlapping test sequences are employed, the primersare designed to hybridize outside (but adjacent) the test sequence.

The term “region” may refer to a relatively large segment of a proteinor an oligonucleotide. Specifically, a region of a protein refers to arange of between 101-1700 contiguous amino acids, while a region of anoligonucleotides refers to a range of between 303-5100 contiguousnucleic acids.

The term “contiguous” when used in reference to a single molecule refersto a continuous, finite, sequence of units wherein each unit hasphysical contact with at least one other unit in the sequence. Forexample, a contiguous sequence of amino acids are physically connectedby peptide bonds and a contiguous sequence of nucleic acids arephysically connect by phosphodiester bonds. When used in the context oftest sequences, contiguous refers to the coverage of the region withoutgaps.

The term “sequence corresponding to a promoter” refers to a non-codingnucleic acid region that is responsible for the regulation oftranscription (an open reading frame) of the DNA coding for the proteinof interest.

The term “sequence corresponding to a ribosome binding site” refers to acoding nucleic acid region that, when transcribed, allows the binding amRNA in such a manner that translation occurs.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. Typically, because ofcurrent limitations of mass spec equipment, an entire protein coded forby a disease gene will not be translated. Nonetheless, the portion maybe referred to as a nascent protein or nascent polypeptide.

The term “wild-type” refers to a gene or gene product (or portionthereof) which has the characteristics of that gene or gene product whenisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” refers to a gene or geneproduct (or portion thereof) which displays modifications in sequenceand or functional properties (i.e., altered characteristics) whencompared to the wild-type gene or gene product. It is noted thatnaturally-occurring mutants can be isolated; these are identified by thefact that they have altered characteristics when compared to thewild-type gene or gene product. Importantly, the mass spec analysis ofmutations does not require functional portions of the protein.

The term “oligonucleotide” as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three, and usually more than ten. The exact sizewill depend on many factors, which in turn depends on the ultimatefunction or use of the oligonucleotide. The oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, or a combination thereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may have 5′ and 3′ ends.

The term “primer” refers to an oligonucleotide which is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically. Primers are used in primer extensionreactions and PCR.

A primer is selected to have on its 3′ end a region that is“substantially” complementary to a strand of specific sequence of thetemplate. A primer must be sufficiently complementary to hybridize witha template strand for primer elongation to occur. A primer sequence neednot reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingsubstantially complementary to the strand. Non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thetemplate to hybridize and thereby form a template primer complex forsynthesis of the extension product of the primer.

As used herein, the terms “hybridize” and “hybridization” refers to theannealing of a complementary sequence to the target nucleic acid. Theability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. Marmur and Lane, Proc. Natl. Acad. Sci.USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461(1960). The terms “annealed” and “hybridized” are used interchangeablythroughout, and are intended to encompass any specific and reproducibleinteraction between an oligonucleotide and a target nucleic acid,including binding of regions having only partial complementarity.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” The term “complement” or“complementary” does not imply or limit pairing to the sense strand orthe antisense strand of a gene; the term is intended to be broad enoughto encompass either situation. Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

The stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m).” The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which on average halfof the base pairs have disassociated. Where amplification is performedin a mixture of genomic DNA, it is convenient to carry out thehybridization of primers at a temperature that is at or above the Tm.

The term “probe” as used herein refers to an oligonucleotide which formsa duplex structure or other complex with a sequence in another nucleicacid, due to complementarity or other means of reproducible attractiveinteraction, of at least one sequence in the probe with a sequence inthe other nucleic acid.

“Oligonucleotide primers matching or complementary to a gene sequence”refers to oligonucleotide primers capable of facilitating thetemplate-dependent synthesis of single or double-stranded nucleic acids.Oligonucleotide primers matching or complementary to a gene sequence maybe used in PCRs, RT-PCRs and the like. As noted above, anoligonucleotide primer need not be perfectly complementary to a targetor template sequence. A primer need only have a sufficient interactionwith the template that it can be extended by template-dependentsynthesis.

As used herein, the term “poly-histidine tract” or (HIS-tag) refers tothe presence of two to ten histidine residues at either the amino- orcarboxy-terminus of a nascent protein A poly-histidine tract of six toten residues is preferred. The poly-histidine tract is also definedfunctionally as being a number of consecutive histidine residues addedto the protein of interest which allows the affinity purification of theresulting protein on a nickel-chelate column, or the identification of aprotein terminus through the interaction with another molecule (e.g. anantibody reactive with the HIS-tag).

As used herein, the term “marker” is used broadly to encompass a varietyof types of molecules (e.g. introduced into proteins using methods andcompositions of the present invention) which are detectable throughspectral properties (e.g. fluorescent markers or “fluorophores”) orthrough functional properties (e.g. affinity markers). An epitope markeror “epitope tag” is a marker of the latter type, functioning as abinding site for antibody or other types of binding molecules (e.g.receptors, lectins and other ligands). Of course, if the epitope markeris used to immobilize the nascent protein, the epitope marker is also anaffinity marker. An epitope has the property that it selectivelyinteracts with molecules and/or materials containing acceptor groups.There are many epitope sequences reported in the literature includingHisX6 (HHHHHH) (SEQ ID NO: 120) described by ClonTech and C-myc(EQKLISEEDL) (SEQ ID NO:122) described by Roche-BM, Flag (DYKDDDDK) (SEQID NO:121) described by Stratagene, SteptTag (WSHPQFEK) (SEQ ID NO:123)described by Sigma-Genosys and HA Tag (YPYDVPDYA) (SEQ ID NO:127)described by Roche-BM. Other epitopes are shown in Table 3 or aredescribed in the examples.

One group of fluorophores with members possessing several favorableproperties (including favorable interactions with components of theprotein translational synthesis system) is the group derived fromdipyrrometheneboron difluoride derivatives (BODIPY). Compared to avariety of other commonly used fluorophores with advantageous propertiessuch as high quantum yields, some BODIPY compounds have the additionalunusual property that they are highly compatible with the proteinsynthesis system. The core structure of all BODIPY fluorophores is4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. See U.S. Pat. Nos.4,774,339; 5,187,288; 5,248,782; 5,274,113; 5,433,896; 5,451,663, allhereby incorporated by reference. A central feature is a difluoroboronas shown in FIG. 4. All BODIPY fluorophores have several desirableproperties for a marker (Molecular Probes Catalog, pages 13-18)including a high extinction coefficient, high fluorescence quantumyield, spectra that are insensitive to solvent polarity and pH, narrowemission bandwidth resulting in a higher peak intensity compared toother dyes such as fluorescein, absence of ionic charge and enhancedphotostability compared to fluorescein. The addition of substituents tothe basic BODIPY structure which cause additional conjugation can beused to shift the wavelength of excitation or emission to convenientwavelengths compatible with the means of detection. An additionaladvantage of BODIPY-FL as a marker is the availability of monoclonalantibodies directed against it which can be used to affinity purifynascent proteins containing said marker. One example of such amonoclonal antibody is anti-BODIPY-FL antibody (Cat# A-5770, MolecularProbes, Eugene, Oreg.). This combined with the ability incorporateBODIPY-FL into nascent proteins with high efficiency relative to othercommercially available markers using misaminoacylated tRNAs facilitatesmore efficient isolation of the nascent protein. These antibodiesagainst BODIPY-FL can be used for quantitation of incorporation of theBODIPY into the nascent protein.

As used herein, the term “total tRNA” is used to describe a mixturecomprising misaminoacylated marker tRNA molecules representing eachamino acid. This mixture has a distinct advantage over the limitedability of misaminoacylated lys-tRNA to reliably incorporate in largevariety of proteins. It is contemplated that “total tRNA” will provide ahomogenous insertion of affinity markers in all nascent proteins.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of (A) an amino acid and (B) (SEO ID NO: 164)a peptide.

FIG. 2 Description of the molecular steps that occur during proteinsynthesis in a cellular or cell-free system (SEQ ID NOS:165-167).

FIG. 3 shows a structure of (A) a tRNA molecule and (B) approachesinvolved in the aminoacylation of tRNAs.

FIG. 4 shows the structure of dipyrrometheneboron difluoride(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.

FIG. 5A is a bar graph showing gel-free quantitation of an N-terminalmarker introduced into a nascent protein in accordance with oneembodiment of the present invention. FIG. 5B is a bar graph showinggel-free quantitation of an C-terminal marker of a nascent proteinquantitated in accordance with one embodiment of the present invention.

FIG. 6 are gel results of in vitro translation results wherein threemarkers were introduced into a nascent protein.

FIG. 7 shows Western blot analysis of in vitro translatedtriple-epitope-tagged wild-type p53 (RT-PCR derived DNA). FIG. 7A showsthe total protein staining. FIG. 7B presents the Western blot analysis.

FIG. 8 shows three bar graphs representing the results of a gel-freechemiluminescent protein truncation assay of p53 and APC. FIG. 8A showsthe results for p53 produced by in vitro translation, where the productis captured in a 96-well ELISA plate format using a mouse monoclonalantibody directed against the N-terminal FLAG epitope. FIG. 8B shows theresults for p53 produced by in vitro translation, where the product iscaptured in a 96-well ELISA plate format using a nickel chelate plate.FIG. 8C shows the results for APC produced by in vitro translation,where the product is captured on nickel metal chelate 96-well ELISAplates. All WT N- and C-terminal signals as well as mutant N-terminalsignals were normalized to 100%.

FIG. 9 shows the effect of addition of cycloheximide on the degradationof the N-terminal (VSV) and C-terminal (P53) signal of nascent protein.

FIG. 10 displays data comparing the signals between standardincorporation of biotin-tRNA^(lys) on an unmodified nascent protein(grey bars) and PCR insertion of five (5) extra terminal lysine residueson a nascent protein (black bars). WT: Wild-Type DNA. N3: HumanTruncated Mutant DNA.

FIG. 11 displays the detection of a point mutation in in vitro expressedalpha-hemolysin by MALDI-TOF. Tracing a: 34,884-WT singly ionizedspecies; [MH]²⁺-WT doubly ionized species. Tracing b: 34,982-mutantsingly ionized species; [MH]²⁺-mutant doubly ionized species.

FIG. 12 shows a time course study wherein a VSV peptide is tested inbuffer and RR extract.

FIG. 13 shows a time course study wherein a P53 peptide is tested inbuffer and RR extract.

FIG. 14 shows a time course study wherein the proteolysis of a VSVpeptide by RR extract is inhibited by a protease inhibitor cocktail.

FIG. 15 shows a time course study wherein the proteolysis of a P53peptide is only partially inhibited by a protease inhibitor cocktail.

FIG. 16 is a bar graph showing the concentration dependence of theprotease inhibitor cocktail on interference with protein production.

FIG. 17 is a bar graph showing that a reconstituted system nonethelesscontains protease activity, albeit less than RR and E. coli extracts.

FIG. 18 is a three-panel readout of mass spec data wherein aprotease-sensitive peptide is shown to be partially degraded at oneminute (middle panel) and completely degraded (lower panel) at fiveminutes by a RR extract, as compared to the control (top).

FIG. 19 is a three-panel readout of mass spec data wherein thedegradation of a protease-sensitive peptide by RR extract is partiallyinhibited by one inhibitor (lower) and only weakly inhibited by anotherinhibitor (middle).

FIG. 20 is a three-panel readout of mass spec data wherein thedegradation of a protease-sensitive peptide by E. coli extract isstrongly inhibited by one inhibitor (middle) and only weakly inhibitedby another inhibitor (lower).

FIG. 21 is a two-panel readout of mass spec data showing thedisappearance of the intact reference peptide (R6) after a 15 minuteexposure to a reconstituted translation system (PURE I) in the absenceof protease inhibitors (top). On other hand, the inhibitor AEBSF showedexcellent inhibition of proteolysis under the same conditions (bottom).

FIG. 22 is a two-panel readout of mass spec data showing virtuallycomplete degradation of the reference peptide (R6) after exposure to thePURE II system with (bottom) and without (top) the AEBSF inhibitor.

FIG. 23 is a seven-panel readout of mass spec data showing that the vastmajority of compounds tested as possible inhibitors in the Pure IIsystem were not effective, with the exception of AEBSF and aprotinin(third and fifth panels).

FIG. 24 is a four-panel readout of mass spec data showing thatinhibiting the proteases in RR extracts (as measured by use of thereference peptide) could not be done with a single compound to a degreenecessary for mass spec analysis.

FIG. 25 is a four-panel readout of mass spec data showing thatinhibiting the proteases in RR extracts (as measured by use of thereference peptide) could not be done with a single compound to a degreenecessary for mass spec analysis, although some inhibition can bemeasured with one inhibitor (top panel).

FIG. 26 is a two-panel readout of mass spec data showing that inhibitingthe proteases in RR extracts (as measured by use of the referencepeptide) could be done with a combination of inhibitors comprisingantipain, aprotinin, calpastatin and α-BOC deacetylleupeptin (the “fourinhibitor cocktail”).

FIG. 27 is a five-panel readout of mass spec data comparing theinhibition of the proteases in RR extracts (as measured by use of thereference peptide) achieved with the combination of the four inhibitorcocktail (bottom) against three inhibitor cocktails. Other combinationswere tested (FIG. 27) and they were either less effective (compare toppanel to bottom panel) or completely ineffective (middle three panels).

FIG. 28 is a readout of mass spec data comparing results with acommercially available reconstituted translation system that has beentreated so as to deplete proteases (FIG. 28B) with the same system thathas not been treated to deplete proteases (FIG. 28A).

FIG. 29 is a readout of mass spec data comparing results whereinwild-type sequences are made together with truncated sequences in an invitro translation system and are either removed by affinitychromatography comprising a ligand to the C-terminal epitope on thewild-type sequences (FIG. 29D) or not removed (FIG. 29C). FIGS. 29A andB are controls wherein wild-type sequences are made alone and truncatedsequences are made alone, respectively.

DESCRIPTION OF THE INVENTION

Nonsense or frameshift mutations, which result in a truncated geneproduct, are prevalent in a variety of disease-related genes. Den Dunnenet al., The Protein Truncation Test: A Review. Hum Mutat 14:95-102(1999). Specifically, these diseases include: i) APC (colorectalcancer), Powell et al., Molecular Diagnosis Of Familial AdenomatousPolyposis. N Engl J Med 329:1982-1987 (1993); van der Luijt et al.,Rapid Detection Of Translation-Terminating Mutations At The AdenomatousPolyposis Coli (APC) Gene By Direct Protein Truncation Test. Genomics20:1-4 (1994); Traverso et al., Detection Of APC Mutations In Fecal DNAFrom Patients With Colorectal Tumors. N Engl J Med 346:311-320 (2002);Kinzler et al., Identification Of A Gene Located At Chromosome 5q21 ThatIs Mutated In Colorectal Cancers. Science 251:1366-1370 (1991); andGroden et al., Identification And Characterization Of The FamilialAdenomatous Polyposis Coli Gene. Cell 66:589-600 (1991); ii) BRCA1 andBRCA2 (breast and ovarian cancer), Hogervosrt et al., Rapid Detection OfBRCA1 Mutations By The Protein Truncation Test. Nat Genet 10:208-212(1995); Garvin et al., A Complete Protein Truncation Test For BRAC1 andBRAC2. Eur J Hum Genet 6:226-234 (1998); Futreal et al., BRAC1 MutationsIn Primary Breast And Ovarian Carcinomas. Science 266:120-122 (1994);iii) polycystic kidney disease, Peral et al., Identification OfMutations In the Duplicated Region Of The Polycystic Kidney Disease 1Gene (PKD1) By A Novel Approach. Am J. Hum Genet 60:1399-1410 (1997);iv) neurofibromatosis (NF1 and NF2), Hein et al., Distribution Of 13Truncating Mutations In The Neurofibromatosis 1 Gene. Hum Mol Genet4:975-981 (1995); Parry et al., Germ-line Mutations In TheNeurofibromatosis 2 Gene: Correlations With Disease Severity And RetinalAbnormalities. Am J Hum Genet 59:529-539 (1996); and v) Duchennemuscular dystrophy (DMD), Roest et al., Protein Truncation Test (PTT) ToRapidly Screen The DMD gene For Translation Terminating Mutations.Neuromuscul Disord 3:391-394 (1993). Such chain truncating mutations canbe detected using the protein truncation test (PTT). This test is basedon cell-free coupled transcription-translation of PCR (RT-PCR) amplifiedportions of the target gene (target mRNA) followed by analysis of thetranslated product(s) for shortened polypeptide fragments. However,conventional PTT is not easily adaptable to high throughput applicationssince it involves SDS-PAGE followed by autoradiography or Western blot.It is also subject to human error since it relies on visual inspectionto detect mobility shifted bands.

To overcome these limitations, we have developed the first highthroughput and high sensitivity truncation test utilizing massspectrometry. This approach uses specially designed PCR primers, whichintroduce N- and C-terminal markers (e.g. epitopes). After translationof the protein fragments, capture and detection is accomplished using aligand which binds the N-terminal epitope. The C-terminal epitope can beused to deplete wild-type sequence. Thereafter, wild-type and truncatedproducts are detected by mass spectrometry.

It was previously not appreciated that small truncation products wouldbe difficult to assess by mass spectrometry because of susceptibility toproteolysis during translation. Even with a reconstituted translationsystem, polypeptides in the 5 to 150 amino acid size range (and inparticular, polypeptides in the 10 to 60 amino acid size range) arequickly degraded, complicating (if not completely preventing) analysis.Since the problem was not recognized, no solutions were explored.Moreover, solving the problem with protease inhibitors is notstraightforward in that a) many inhibitors do not inhibit, and b) someinhibitors that inhibit proteolysis also inhibit translation. In oneembodiment, the present invention provides, on the other hand, proteaseinhibitors (single compounds for the reconstituted systems andcombinations for other systems) which do not significantly interferewith translation at the levels needed to inhibit proteolysis. In anotherembodiment, components in the reconstituted system are manipulated so asto reduce proteases in the mixture. In another embodiment, combinationsof these approaches can be utilized.

Thus, in one embodiment where one or more components are manipulated,the present invention contemplates a method, comprising: a) providing i)a cell-free translation system comprising a first preparation comprisingfirst ribosomes in solution, and ii) second ribosomes; b) removing atleast a portion of said first ribosomes from said solution (e.g. byfiltration, centrifugation, precipitation, etc.) so as to create adepleted solution; c) adding second ribosomes to said depleted solutionso as to create a second preparation, wherein the protease activity ofsaid first preparation is greater than the protease activity of saidsecond preparation. In a preferred embodiment, said second ribosomes areribosomes purified by zonal centrifugation. It is particularly preferredthat protease activity of said first and second preparations is measuredusing mass spectrometry, such as the mass spec-based protease assaydescribed herein which employs a (non-naturally occurring)protease-sensitive peptide.

Regardless of the approach, the present invention contemplates in oneembodiment a method, comprising: providing: i) a nucleic acid sequenceencoding a polypeptide, said polypeptide being between 10 and 150 aminoacids in length (and more preferrably between 10 and 100 amino acids inlength, and still more preferrably between 10 and 80 amino acids inlength); and ii) an in vitro translation system; and introducing saidnucleic acid into said translation system under conditions such thatsaid polypeptide is produced, wherein said polypeptide is degraded byless than 50% (and more preferrably by less than 30%) by proteolysisfollowing exposure to said translation system for approximately 10minutes (and more preferrably, for 20 minutes) at approximately 37° C.In one embodiment, the present invention contemplates that proteolysisis measured by mass spectrometry. In one embodiment, said nucleic isDNA; in another embodiment, it is RNA (e.g. RNA made by in vitrotranscription from a PCR product, such as a PCR product amplified fromgenomic DNA obtained from a whole organism, including humans). In apreferred embodiment, said nucleic acid sequence comprises a portioncomplementary to a portion of a disease-related gene (e.g. the APCgene).

Regardless of the approach, the present invention contemplates inanother embodiment a method, comprising: providing: i) a nucleic acidsequence encoding a polypeptide, said polypeptide being between 10 and150 amino acids in length (and more preferrably between 10 and 100 aminoacids in length, and still more preferrably between 10 and 80 aminoacids in length); and ii) an in vitro translation system that has beentreated to reduce protease activity such that a protease-sensitivereference peptide is degraded by less than 50% (and more preferrably byless than 30%) by proteolysis following exposure to said translationsystem for approximately 10 minutes (and more preferrably, for 20minutes) at approximately 37° C.; and introducing said nucleic acid intosaid translation system under conditions such that said polypeptide isproduced. In one embodiment, the present invention contemplates thatproteolysis is measured by mass spectrometry. In one embodiment, saidnucleic is DNA; in another embodiment, it is RNA (e.g. RNA made by invitro transcription from a PCR product, such as a PCR product amplifiedfrom genomic DNA obtained from a whole organism, including humans). In apreferred embodiment, said nucleic acid sequence comprises a portioncomplementary to a portion of a disease-related gene (e.g. the APCgene).

Regardless of the approach, the present invention contemplates in yetanother embodiment a method, comprising: providing a preparationcomprising polypeptides, said polypeptides being between 10 and 150amino acids in length (and more preferrably, between 10 and 100 aminoacids in length, and still more preferrably, between 10 and 80 aminoacids in length) and comprising a C-terminal epitope; and determiningthe molecular mass of said polypeptides by mass spectrometry. In aparticularly preferred embodiment, said polypeptides further comprise anN-terminal epitope. In a preferred embodiment, said wild typepolypeptides were made by in vitro translation (e.g. using a cell-freetranslation system), and in particular, a translation system that hasbeen treated under conditions such that the protease activity is reduced(e.g. to a level such that there is less than 50% degradation of nascentpolypeptides of the stated size range after exposure for 10 minutes at37 degrees). Moreover, while a variety of proteins can be evaluated inthis manner, the present invention contemplates a preferred embodimentwherein at least a portion of each of said polypeptides is identical toa portion of the disease related gene product (e.g. APC gene product).

The mass spectrometry approach is generally applicable and is hereindemonstrated for the detection of chain truncation mutations in the APCgene. It is readily applied to the DNA of individuals pre-diagnosed withfamilial adenomatous polyposis (FAP). This mass spec approach provides ahigh throughput method for non-invasive colorectal cancer screening.Importantly, there is no need to enrich for low-abundance mutant DNA(although in preferred embodiment, one may enrich for low abundancetruncated polypeptides by depleting wild-type sequences).

As embodied and described herein, the present invention comprisesmethods for the labeling the products of new or nascent proteinsynthesis, and methods for the isolation of these nascent proteins frompreexisting proteins in a cellular or cell-free translation system sothat detection can be performed by mass spectrometry.

While the preferred use of the invention is to detect truncationmutations, any proteins that can be expressed by translation in acellular or cell-free translation system can be evaluated as nascentproteins and consequently, labeled, detected and isolated by the methodsof the invention. Examples of such proteins include enzymes such asproteolytic proteins, cytokines, hormones, immunogenic proteins,carbohydrate or lipid binding proteins, nucleic acid binding proteins,human proteins, viral proteins, bacterial proteins, parasitic proteinsand fragments and combinations. These methods are well adapted for thedetection of products of recombinant genes and gene fusion productsbecause recombinant vectors carrying such genes generally carry strongpromoters which transcribe mRNAs at fairly high levels. These mRNAs areeasily translated in a translation system.

Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include whole cellpreparations such as permeabilized cells or cell cultures wherein adesired nucleic acid sequence can be transcribed to mRNA and the mRNAtranslated.

Cell-free translation systems are commercially available and manydifferent types and systems are well-known. Examples of cell-freesystems include prokaryotic lysates such as Escherichia coli lysates,and eukaryotic lysates such as wheat germ extracts, insect cell lysates,rabbit reticulocyte lysates, frog oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified becausemany such modifications are only possible in eukaryotic systems. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Less than 10 nanoliters of acommercially available E. coli extract (E. coli T7 translation system,Promega, Madison, Wis.) are needed for analysis corresponding to lessthan 1 ng of synthesized protein. Membranous extracts, such as thecanine pancreatic extracts containing microsomal membranes, are alsoavailable which are useful for translating secretory proteins. Mixturesof purified translation factors have also been used successfully totranslate mRNA into protein as well as combinations of lysates orlysates supplemented with purified translation factors such asinitiation factor-1 (IF-1), IF-2, IF-3, elongation factor T (EF-Tu), ortermination factors.

A preferred translation system is a reconstituted system available fromPost Genome Institute Co., Ltd. (Japan) called PURESYSTEM. The systemswas originally developed at the University of Tokyo and comprisesapproximately 30 purified enzymes necessary for transcription andtranslation. Because all the components are tagged with a hexahistidine,the preferred N-terminal and C-terminal epitopes for the wild-type andtruncated polypeptides (discussed in various embodiments of the methodbelow) are preferably not Histags. The system is advertised as“essentially free of protease,” however, there is significant proteaseactivity that interferes with detection of small polypeptides by massspectrometry. In one embodiment, the present invention contemplatessupplementing a reconstituted system with a protease inhibitor. Forexample, in one embodiment, the present invention contemplates acell-free translation system comprising a) ribosomes; b) recombinantlyproduced proteins, said proteins comprising one or more initiationfactors, one or more elongation factors, one or more release factors, aplurality of aminoacyl-tRNA synthetases, and methionyl-tRNAtransformylase; and c) one or more protease inhibitors. In oneembodiment, said protease inhibitor is aprotinin. In a preferredembodiment, said protease inhibitor is used at a concentration thatinhibits proteolysis but does not significantly interfere withtranslation (e.g. yield of nascent protein is reduced by less than 50%in a 20 minute reaction at 37 degrees, and more preferrably, reduced byless than 30%). In a particularly preferred embodiment, said proteaseinhibitor is AEBSF.

In one embodiment, the present invention contemplates a cell-freetranslation system comprising a) ribosomes; b) recombinantly producedproteins, said proteins comprising one or more initiation factors, oneor more elongation factors, one or more release factors, a plurality ofaminoacyl-tRNA synthetases, and methionyl-tRNA transformylase; and c) aprotease-sensitive peptide. In one embodiment, the protease-sensitivepeptide is chemically synthesized. However, in another embodiment, theprotease-sensitive peptide is made by the translation system. In oneembodiment, the present invention contemplates a kit, comprising: a)ribosomes; b) recombinantly produced proteins comprising one or moreinitiation factors, one or more elongation factors, one or more releasefactors, a plurality of aminoacyl-tRNA synthetases, and methionyl-tRNAtransformylase; and c) a chemically synthesized, protease-sensitivepeptide. In another embodiment, the present invention contemplates akit, comprising: a) ribosomes; b) recombinantly produced proteinscomprising one or more initiation factors, one or more elongationfactors, one or more release factors, a plurality of aminoacyl-tRNAsynthetases, and methionyl-tRNA transformylase; and c) a nucleic acidencoding a protease-sensitive peptide.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a nucleic acid sequence encoding apolypeptide, said polypeptide being between 10 and 150 amino acids inlength (and more preferrably between 10 and 100 amino acids in length,and even more preferrably between 10 and 80 amino acids in length); andii) a reconstituted in vitro translation system comprising recombinantproteins (e.g. purified proteins necessary for in vitro transcriptionand translation); b) introducing said nucleic acid into saidreconstituted translation system under conditions such that saidpolypeptide is produced; and c) determining the molecular mass of saidpolypeptide by mass spectrometry. In one embodiment, said nucleic acidis DNA; in another embodiment, it is RNA (e.g. RNA made by in vitrotranscription from a PCR product, such as a PCR product amplified fromgenomic DNA obtained from a whole organism, including humans). In apreferred embodiment, said nucleic acid sequence comprises a portioncomplementary to a portion of a disease-related gene (e.g. the APCgene). In one embodiment, the reconstituted translation system has beensupplemented with a protease inhibitor. In another embodiment, thereconstituted translation system has been treated to deplete proteases(e.g. by filtering and/or replacing with highly purified ribosomes). Inone embodiment, the polypeptide produced has both an N-terminal andC-terminal epitope and is separated (e.g. prior to step c) from saidtranslation system by a ligand to one of the epitopes (or ligands toboth epitopes). In a preferred embodiment, the polypeptide is separatedby a ligand to the N-terminal epitope.

Regardless of the approach, the present invention contemplates in oneembodiment a cell-free translation system comprising a) ribosomes; andb) recombinantly produced proteins, said proteins comprising one or moreinitiation factors, one or more elongation factors, one or more releasefactors, a plurality of aminoacyl-tRNA synthetases, and methionyl-tRNAtransformylase, wherein said ribosomes and said recombinantly producedproteins in a mixture exhibit protease activity at a level such that aprotease-sensitive reference peptide of between 10 and 30 amino acids inlength is degraded by less than 50% (and preferrably less than 40%, andmore preferrably less than 30%, and still more preferrably less than20%) following exposure to said mixture for approximately 20 minutes atapproximately 37° C. While desirable, it is not required that allprotease activity be eliminated or inhibited.

The present invention further contemplates in one embodiment a cell-freetranslation system comprising a) a protease inhibitor, b) ribosomes; andc) recombinantly produced proteins, said proteins comprising one or moreinitiation factors, one or more elongation factors, one or more releasefactors, a plurality of aminoacyl-tRNA synthetases, and methionyl-tRNAtransformylase, wherein said ribosomes and said recombinantly producedproteins in a mixture with said protease inhibitor exhibit proteaseactivity at a level such that a protease-sensitive reference peptide ofbetween 10 and 30 amino acids in length is degraded by less than 50%(and preferrably less than 40%, and more preferrably less than 30%, andstill more preferrably less than 20%) following exposure to said mixturefor approximately 20 minutes at approximately 37° C. While desirable, itis not required that all protease activity be eliminated or inhibited.

In one embodiment, the present invention contemplates a method,comprising: a) providing: i) a nucleic acid sequence encoding aprotease-sensitive reference polypeptide, said polypeptide being between5 and 50 amino acids in length (and more preferrably between 10 and 30amino acids in length, and even more preferrably between 15 and 25 aminoacids in length); and ii) a reconstituted in vitro translation systemcomprising recombinant proteins (e.g. purified proteins necessary for invitro transcription and translation); and b) introducing said nucleicacid into said reconstituted translation system under conditions suchthat said polypeptide is produced. In a preferred embodiment, the methodfurther comprises c) detecting the polypeptide by mass spectrometry. Ina particularly preferred embodiment, the method further comprises c)detecting the proteolytic degradation of said polypeptide by massspectrometry. In one embodiment, n the protease-sensitive peptidecomprises an N-terminal epitope (for convenient capture and purificationfrom the mixture). In a preferred embodiment, the protease-sensitivepeptide further comprises a region of positively charged amino acidsselected from the group consisting of arginine, lysine or histidine. Ina more preferred embodiment, the protease-sensitive peptide furthercomprises a C-terminal region comprising hydrophobic amino acids (e.g.phenylalanine). Optionally, the N-terminus can have other amino acids(e.g. methionine) or protecting groups (FMOC, etc.). In one embodiment,the protease-sensitive reference polypeptide is selected from thepolypeptides set forth in Table 2.

The PURESYSTEM lacks tRNAs to rare codons. In one embodiment, thepresent invention contemplates supplementing a reconstituted system withtRNAs to rare codons For example, in one embodiment, the presentinvention contemplates a cell-free translation system comprising a)ribosomes; b) recombinantly produced proteins, said proteins comprisingone or more initiation factors, one or more elongation factors, and oneor more release factors, a plurality of aminoacyl-tRNA synthetases, andmethionyl-tRNA transformylase; and c) a plurality of tRNAs, said tRNAscomprising tRNAs for one or more codons selected from the groupconsisting of AGG, AGA, AUA, CUA, CCC and GGA. In one embodiment, thepresent invention contemplates a kit, comprising a) ribosomes; b)recombinantly produced proteins, said proteins comprising one or moreinitiation factors, one or more elongation factors, one or more releasefactors, a plurality of aminoacyl-tRNA synthetases, and methionyl-tRNAtransformylase; and c) a plurality of tRNAs, said tRNAs comprising tRNAsfor one or more codons selected from the group consisting of AGG, AGA,AUA, CUA, CCC and GGA. Such a kit may further comprise instructions forcarrying out translation.

Cell-free systems may also be coupled transcription/translation systemswherein DNA is introduced to the system, transcribed into mRNA and themRNA translated as described in Current Protocols in Molecular Biology(F. M. Ausubel et al. editors, Wiley Interscience, 1993), which ishereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

tRNA molecules chosen for misaminoacylation with marker do notnecessarily possess any special properties other than the ability tofunction in the protein synthesis system. Due to the universality of theprotein translation system in living systems, a large number of tRNAscan be used with both cellular and cell-free reaction mixtures. SpecifictRNA molecules which recognize unique codons, such as nonsense or ambercodons (UAG), can be used but are not required in all embodiments.

Site-directed incorporation of the nonnative analogs into the proteinduring translation is also not required. Incorporation of markers canoccur anywhere in the polypeptide and can also occur at multiplelocations. This eliminates the need for prior information about thegenetic sequence of the translated mRNA or the need for modifying thisgenetic sequence.

tRNAs molecules used for aminoacylation are commercially available froma number of sources and can be prepared using well-known methods fromsources including Escherichia coli, yeast, calf liver and wheat germcells (Sigma Chemical; St. Louis, Mo.; Promega; Madison, Wis.;Boehringer Mannheim Biochemicals; Indianapolis, Ind.). Their isolationand purification mainly involves cell-lysis, phenol extraction followedby chromatography on DEAE-cellulose. Amino-acid specific tRNA, forexample tRNA^(fMet), can be isolated by expression from cloned genes andoverexpressed in host cells and separated from total tRNA by techniquessuch as preparative polyacrylamide gel electrophoresis followed by bandexcision and elution in high yield and purity (Seong and RajBhandary,Proc. Natl. Acad. Sci. USA 84:334-338, 1987). Run-off transcriptionallows for the production of any specific tRNA in high purity, but itsapplications can be limited due to lack of post-transcriptionalmodifications (Bruce and Uhlenbeck, Biochemistry 21:3921, 1982).

In the cell-free protein synthesis system, the reaction mixture containsall the cellular components necessary to support protein synthesisincluding ribosomes, tRNA, rRNA, spermidine and physiological ions suchas magnesium and potassium at appropriate concentrations and anappropriate pH. Reaction mixtures can be normally derived from a numberof different sources including wheat germ, E. coli (S-30), red bloodcells (reticulocyte lysate) and oocytes, and once created can be storedas aliquots at about +4° C. to −70° C. The method of preparing suchreaction mixtures is described by J. M. Pratt (Transcription andTranslation, B. D. Hames and S. J. Higgins, Editors, p. 209, IRL Press,Oxford, 1984) which is hereby incorporated by reference. Many differenttranslation systems are commercially available from a number ofmanufacturers.

Translations in cell-free systems generally require incubation of theingredients for a period of time. Incubation times range from about 5minutes to many hours, but is preferably between about ten and thirtyminutes and more preferably between about ten to twenty minutes.Incubation may also be performed in a continuous manner whereby reagentsare flowed into the system and nascent proteins removed or left toaccumulate using a continuous flow system (A. S. Spirin et al., Sci.242:1162-64, 1988). This process may be desirable for large scaleproduction of nascent proteins. Incubations may also be performed usinga dialysis system where consumable reagents are available for thetranslation system in an outer reservoir which is separated from largercomponents of the translation system by a dialysis membrane [Kim, D.,and Choi, C. (1996) Biotechnol Prog 12, 645-649]. Incubation times varysignificantly with the volume of the translation mix and the temperatureof the incubation. Incubation temperatures can be between about 4° C. toabout 60° C., and are preferably between about 15° C. to about 50° C.,and more preferably between about 25° C. to about 45° C. and even morepreferably at about 25° C. or about 37° C. Certain markers may besensitive to temperature fluctuations and in such cases, it ispreferable to conduct those incubations in the non-sensitive ranges.Translation mixes will typically comprise buffers such as Tris-HCl,Hepes or another suitable buffering agent to maintain the pH of thesolution between about 6 to 8, and preferably at about 7. Other reagentswhich may be in the translation system include dithiothreitol (DTT) or2-mercaptoethanol as reducing agents, RNasin to inhibit RNA breakdown,and nucleoside triphosphates or creatine phosphate and creatine kinaseto provide chemical energy for the translation process.

In cellular protein synthesis, it is necessary to introduce RNA or DNAinto intact cells, cell organelles, cell envelopes and other discretevolumes bounded by an intact biological membrane, which contain aprotein synthesizing system. This can be accomplished through a varietyof methods that have been previously established such as sealing thetRNA solution into liposomes or vesicles which have the characteristicthat they can be induced to fuse with cells. Fusion introduces theliposome or vesicle interior solution containing the tRNA into the cell.Alternatively, some cells will actively incorporate liposomes into theirinterior cytoplasm through phagocytosis. The tRNA solution could also beintroduced through the process of cationic detergent mediatedlipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-17,1987), or injected into large cells such as oocytes. Injection may bethrough direct perfusion with micropipettes or through the method ofelectroporation.

Alternatively, cells can be permeabilized by incubation for a shortperiod of time in a solution containing low concentrations of detergentsin a hypotonic media. Useful detergents include Nonidet-P 40 (NP40),Triton X-100 (TX-100) or deoxycholate at concentrations of about 0.01 nMto 1.0 mM, preferably between about 0.1 M to about 0.01 mM, and morepreferably about 1 M. Permeabilized cells allow marker to pass throughcellular membranes unaltered and be incorporated into nascent proteinsby host cell enzymes. Such systems can be formed from intact cells inculture such as bacterial cells, primary cells, immortalized cell lines,human cells or mixed cell populations. These cells may, for example, betransfected with an appropriate vector containing the gene of interest,under the control of a strong and possibly regulated promoter. Messagesare expressed from these vectors and subsequently translated withincells. Intact misaminoacylated tRNA molecules, already charged with anon-radioactive marker could be introduced to cells and incorporatedinto translated product.

It will be understood by those skilled in the area of molecular biologyand biochemistry that the N-terminal marker, affinity marker andC-terminal marker can all consist of epitopes that can be incorporatedinto the nascent protein by designing the message or DNA coding for thenascent protein to have a nucleic acid sequence corresponding to theparticular epitope. This can be accomplished using known methods such asthe design of primers that incorporate the desired nucleic acid sequenceinto the DNA coding for the nascent protein using the polymerase chainreaction (PCR).

For optimal effectiveness, the N-terminal marker and affinity markershould be placed as close as possible to the N-terminal end of theprotein. For example, if an N-terminal marker is incorporated using amisaminoacylated initiator, it will be located at the N-terminal aminoacid. In this case, the affinity marker should be located immediatelyadjacent to the N-terminal marker. For optimal effectiveness, theC-terminal marker should be placed as close as possible to theC-terminal end of protein. For example, if a His-X6 tag is utilized, theprotein sequence would terminate with 6 His. In some cases, an epitopemay be located several residues before the C-terminal end of the proteinin order to optimize the properties of the nascent protein. This mightoccur for example, if a specific amino acid sequence is necessary inorder to modify specific properties of the nascent protein that aredesirable such as its solubility, hydrophobicity and ability to ionize.

There are several unique advantages of this method compared to existingtechniques for detecting chain terminating or out-of-frame mutations.Normally, such mutations are detected by analyzing the entire sequenceof the suspect gene using conventional DNA sequencing methods. However,such methods are time consuming, expensive and not suitable for rapidthroughput assays of large number of samples. An alternative method isto utilize mass spec, which is able to detect changes from the expectedsize of a nascent protein.

Detecting a protein with an epitope located near the C-terminal end ofthe protein provides information about the presence of either aframeshift or chain terminating mutation since the presence of eitherwould result in an incorrect sequence. The measurement of the N-terminalmarker provides an internal control to which measurement of theC-terminal marker can be normalized. Separating the protein from thetranslation mixture using an affinity marker located at or close to theN-terminal end of the protein eliminates the occurrence of false startswhich can occur when the protein is initiated during translation from aninternal AUG in the coding region of the message. A false start can leadto erroneous results since it can occurs after the chain terminating orout-of-frame mutation. This is especially true if the internal AUG isin-frame with the message. In this case, the peptide C-terminal markerwill still be present even if message contains a mutation.

DETAILED DESCRIPTION

Mass spectrometry measures the mass of a molecule. The use of massspectrometry in biology is continuing to advance rapidly, findingapplications in diverse areas including the analysis of carbohydrates,proteins, nucleic acids and biomolecular complexes. For example, thedevelopment of matrix assisted laser desorption ionization (MALDI) massspectrometry (MS) has provided an important tool for the analysis ofbiomolecules, including proteins, oligonucleotides, andoligosacharrides. Thus far, it has been found applicable in diverseareas of biology and medicine including the rapid sequencing of DNA,screening for bioactive peptides and analysis of membrane proteins.

Markers introduced into nascent proteins, especially at a specificposition such at the N-terminal, can be used to isolate the proteinprior to the detection by mass spectrometry. Without such a marker, itcan be very difficult to detect a peak because a nascent proteinsynthesized in the presence of a cellular or cell-free extract is in thepresence of many other molecules of similar mass in the extract. Forexample, in some cases less than 0.01% of the total protein mass of theextract may comprise the nascent protein(s). Furthermore, molecules withsimilar molecular weight as the nascent protein may be present in themixture. Such molecules will overlap with peaks due to the nascentprotein. This problem is particularly severe if the nascent protein is atranscription or translation factor already present in the cell-free orcellular protein synthesis. The synthesis of additional amounts of thisprotein in the protein synthesis system would be difficult to detectusing known methods in mass spectrometry since peak intensities are notcorrelated in a linear manner with protein concentration.

The sensitivity of mass spec creates unique issues. While the proteaseconcentration in commercially available translation systems may not beproblematic for some applications, the presence of protease in thecontext of mass spectrometry can completely obscure detection.Consequently, preferred embodiments of the invention employ means toreduce and/or eliminate proteolysis of in vitro (cell-free) expressedproteins and protein fragments which are used specifically fordiagnostic purposes such as described herein. These means can includebut are not limited to the addition of compounds to the in vitro proteinexpression system which inhibit the proteolytic processes, theelimination of factors from the mixture which are involved inproteolysis, the inactivation of factors through physical meansincluding heating, light and physical binding to other molecules, thedesign of expressed polypeptide sequences which are resistant toproteolysis and the incorporation into the polypeptide of non-nativeamino acids which increase resistance to proteolysis includingmodifications on the N-terminal and C-terminal end of the polypeptide.

Although, up to now the role that proteolysis may play in using in vitroexpressed proteins for diagnostic purposes has not been emphasized, wehave performed experiments that demonstrate that such proteolyticprocesses can hinder the use of in vitro expressed proteins and proteinfragments for such purposes. For example, many of the methods describedherein involve the in vitro expression of a protein or protein fragmentfrom a DNA or mRNA template followed by its isolation and/or detectionusing specific epitopes which are recognized by specific antibodies orby the incorporation of non-native amino acids through the use ofmis-aminoacyltated tRNAs that subsequently react with a binding moleculesuch as a combination of biotin and streptavidin. In all of the abovecases, proteolysis of the protein or protein fragment can interfere withisolation and/or detection steps.

DESCRIPTION OF PREFERRED EMBODIMENTS Colorectal Cancer Detection

The present invention contemplates the isolation, detection andidentification of expressed proteins having an altered primary aminoacid sequence. One example of an altered primary sequence is a proteinchain truncation. A protein chain truncation is most easily explained bya frameshift mutation that generates a stop codon (i.e., AUG) within theopen reading frame. The resulting translation of the mRNA from thismutated gene synthesizes a nonfunctional or malfunctional protein. Oneexample of such a truncated protein is derived from the APC gene, and isknown to be a diagnostic marker for colorectal cancer.

Many attempts have been reported to detect and analyze biologicalsamples using a noninvasive diagnostic marker of colorectal cancer.Currently, the most reliable method to identify and treat colorectalcancer requires a colonoscopy. While colonoscopy is not a high riskprocedure, except for the associated general anesthesia, it is expensiveand there is a serious problem regarding obtaining compliance for onetime or repeated testing due to the invasive nature of the examinationand the extensive bowel preparation required. One possible non-invasivesource of diagnostic markers is fecal matter.

The Protein Truncation Test (PTT) was first reported by Roest et al.,Protein Truncation Test (PTT) For Rapid Detection OfTranslation-Terminating Mutations. Hum Mol Genet 2:1719-1721 (1993), andapplied to the detection of truncating mutations in the APC gene byPowell et al., Molecular Diagnosis Of Familial Adenomatous Polyposis. N.Engl J Med 329:1982-1987 (1993). In traditional PTT, the region of thegene to be analyzed is amplified by PCR (or RT-PCR for an mRNA template)using a primer pair that incorporates additional sequences into the PCRamplicons required for efficient cell-free translation. The amplifiedDNA is then added to a cell-free transcription-translation extract alongwith radioactive amino acids (³⁵S-methionine or ¹⁴C-leucine). Theexpressed protein is analyzed by SDS-PAGE and autoradiography. Chaintruncation mutations are detected by the presence of a lower molecularweight (increased mobility) species relative to the wild-type (WT)protein band. Non-radioactive Western blot-based PTT-methods utilizing acombination of N-terminal and C-terminal epitopes have also beenreported. Rowan et al., Introduction Of A myc Reporter Tag To ImproveThe Quality Of Mutation Detection Using The Protein Truncation Test. HumMutat 9:172-176 (1997); de Koning Gans et al., A Protein Truncation TestFor Emery-Dreifuss Muscular Dystrophy (EMU): Detection Of N-TerminalTruncating Mutations. Neuromuscul Disord 9:247-250 (1999); and Kahamnnet al., A Non-Radioactive Protein Truncation Test For The SensitiveDetection Of All Stop And Frameshift Mutations. Hum Mutat 19:165-172(2002). However, these approaches still involve lengthy steps ofSDS-PAGE, electroblotting and membrane-based immunoassay.

As an alternative to SDS-PAGE based PTT, the present inventioncontemplates a high throughput mass spec approach. Amplified DNAcorresponding to the region of interest in the target gene is firstgenerated using PCR with primers that incorporate N- and C-terminalepitope tags as well as a T7 promoter, Kozak sequence and start codon(ATG) in the amplicons. The resulting amplified DNA is subsequentlyadded to a cell-free protein expression system (preferably areconstituted system, including but not limited to a reconstitutedsystem that has been further treated or modified to reduce proteaseactivity). The N-terminal epitope is used to capture the translatedprotein from the cell-free expression mixture onto a solid surface. TheC-terminal epitope tag can be used to deplete wild-type sequences.

As an initial evaluation, this mass spec approach was used to detecttruncating mutations in a region of the APC gene (segment 3; amino acids1098-1696) using genomic DNA as a PCR template. While various epitopetag sequences including His-6, c-myc, P53 (derived from the P53sequence), VSV-G, Fil-16 (filamin derived) and StrepTag can be used,FLAG and HA were chosen (in one embodiment) as the N- and C-terminalepitopes, respectively. In order to enhance throughput, the nascentfragment of APC segment 3 was selectively captured from the reactionmixture via the N-terminal tag, thereby conveniently separating thenascent protein from the translation system. To enhance sensitivity,wild-type sequences with a C-terminal epitope can be depleted (prior tomass spec) by exposure to a ligand having affinity for the C-terminalepitope.

DNA derived from patients with familial adenomatous polyposis (FAP) aswell as cell-line DNA with known mutations in segment 3 can be analyzed.

While heterozygous mutations in germ-line cells are expected to comprise50% of the total DNA in a sample, sporadic mutations are often presentin significantly lower abundance, such as the case of stool samples fromindividuals with colorectal cancer. Traverso et al., Detection Of APCMutations In Fecal DNA From Patients With Colorectal Tumors. N Engl J.Med 346:311-320 (2002); Deuter et al., Detection Of APC Mutations InStool DNA Of Patients With Colorectal Cancer By HD-PCR. Hum Mutat,11:84-89 (1998); and Doolittle et al., Detection Of The Mutated K-RasBiomarker In Colorectal Carcinoma. Exp Mol Pathol 70:289-301 (2001). Oneapproach, termed digital PTT, has been utilized to overcome thisproblem. However, the mass spec approach described here does not requiresuch serial dilution of DNA prior to PCR amplification.

Using the mass spectrometry approach described herein, detection of amutation can be made wherein the mutated copies of the gene are presentin a ratio of 1:250 (vis-à-vis the wild type sequences). At thepolypeptide level, detection of a truncated polypeptide can be made in aratio of 1:100 (but more routinely in a ratio of 1:50, as still moreroutinely in a ration of 1:10, vis-à-vis the wild type polypeptide).

The present invention contemplates the isolation, detection andidentification of mutated genes by methods that do not require extensiveand expensive purification, isolation and sequencing procedures.Furthermore, the present invention contemplates the use of nucleic acidmaterial from any tissue or fluid sample, and is not restricted to fecalsamples. Specifically, sample DNA from a patient suspected of havingcancer is amplified by PCR using primers comprising sequences encoding aN-terminal and C-terminal epitope. The epitope-containing sample DNA isplaced in a translation system (i.e., resulting in the production ofmRNA followed by protein synthesis).

Experimental

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention. In some ofthe examples below, particular reagents and methods were employed asfollows:

Reagents: tRNA^(fmet), aminoacyl-tRNA synthetase, amino acids, buffersalts, and RNase free water were purchased from Sigma (St. Louis, Mo.).Many of the fluorescent dyes were obtained from Molecular Probes(Eugene, Oreg.). The translation supplies including routine kits werepurchased from Promega (Madison, Wis.). Sephadex G-25 was fromAmersham-Pharmacia Biotech (Piscataway, N.J.). The in vitro translationkits and plasmid DNAs coding for CAT (PinPoint™) and Luciferase(pBESTluc™) were from Promega (Wisconsin-Madison, Wis.) while DHFRplasmid DNA (pQE16-DHFR) was obtained from Qiagen (Valencia, Calif.).The plasmid DNA for alpha-hemolysin, pT7-WT-H6-HL was kindly supplied byProf. Hagan Bayley (Texas A &M University) and large scale preparationof alpha-HL DNA was carried out using Qiagen plasmid isolation kit. Thebacterioopsin plasmid DNA (pKKbop) was from the laboratory stock.

Preparation of FluoroTag tRNAs: The purified tRNA^(fmet) was firstaminoacylated with the methionine. In typical reaction, 1500 picomoles(˜1.0 OD₂₆₀) of tRNA was incubated for 45 min at 37 C in aminoacylationmix using excess of aminoacyl tRNA-synthetases. After incubation, themixture was neutralized by adding 0.1 volume of 3 M sodium acetate, pH5.0 and subjected to chloroform:acid phenol extraction (1:1). Ethanol(2.5 volumes) was added to the aqueous phase and the tRNA pelletobtained was dissolved in the water (25 ul). The coupling ofNHS-derivatives of fluorescent molecules to the alpha-amino group ofmethionine was carried out in 50 mM sodium carbonate, pH 8.5 byincubating the aminoacylated tRNAf^(met)(25 ul) with fluorescent reagent(final concentration=2 mM) for 10 min at 0 C and the reaction wasquenched by the addition of lysine (final concentration=100 mM). Themodified tRNA was precipitated with ethanol and passed through SephadexG-25 gel filtration column (0.5×5 cm) to remove any free fluorescentreagent, if present. The modified tRNA was stored frozen (−70 C) insmall aliquots in order to avoid free-thaws. The modification extent ofthe aminoacylated-tRNA was assessed by acid-urea gel electrophoresis.This tRNA was found to stable at least for 6 month if stored properly.

Cell free synthesis of proteins and their detection: The in vitrotranslation reactions were typically carried out using E. coli T7transcription-translation system (Promega) with optimized premix. Thetypical translation reaction mixture (10 ul) contained 3 ul of extract,4 ul of premix, 1 ul of complete amino acid mix, 30 picomoles offluorescent-methionyl-tRNA and 0.5 ug of appropriate plasmid DNA. Theoptimized premix (1×) contains 57 mM HEPES, pH 8.2, 36 mM ammoniumacetate, 210 mM potassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mMATP, 0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6mM cAMP and 16 mM magnesium acetate. The translation reaction wasallowed to proceed for 45 min at 37 C. For SDS-PAGE, 4-10 ul aliquot ofthe reaction mix was precipitated with 5-volume acetone and theprecipitated proteins were collected by centrifugation. The pellet wasdissolved in 1× loading buffer and subjected to SDS-PAGE after boilingfor 5 min. SDS-PAGE was carried out according to Laemmli and the gel wasscan using Molecular Dynamics FluorImager 595 using Argon laser asexcitation source. Alternatively, the nascent proteins in polyacrylamidegels were also detected using an UV-transilluminator and the photographswere carried out using Polaroid camera fitted with green filter (Tiffengreen #58, Polaroid DS34 camera filter kit).

For visualization of BODIPY-FL labeled protein, 488 nm as excitationsource was used along with a 530±30 narrow band excitation filter. Thegel was scanned using PMT voltage 1000 volts and either 100 or 200micron pixel size.

Enzyme/Protein activities: Biological activity of alpha-hemolysin wascarried out as follows. Briefly, various aliquots (0.5-2 ul) of in vitrotranslation reaction mixture were added to 500 ul of TBSA (Tris-bufferedsaline containing 1 mg/ml BSA, pH 7.5). To this, 25 ul of 10% solutionof rabbit red blood cells (rRBCs) was added and incubated at roomtemperature for 20 min. After incubation, the assay mix was centrifugedfor 1 min and the absorbance of supernatant was measured at 415 nm(release of hemoglobin). The equal amount of rRBCs incubated in 500 ulof TBSA is taken as control while rRBCs incubated with 500 ul of wateras taken 100% lysis. The DHFR activity was measuredspectrophotometrically. Luciferase activity was determined usingluciferase assay system (Promega) and luminescence was measures usingPackard Lumi-96 luminometer.

Purification of alpha-HL and measurement BODIPY-FL incorporation intoalpha-HL: The translation of plasmid coding for alpha-HL (His₆) wascarried out at 100 ul scale and the alpha-HL produced was purified usingTalon-Sepharose (ClonTech) according manufacturer instructions. Thefluorescence incorporated into alpha-HL was then measured on MolecularDynamics FluorImager along with the several known concentration of freeBODIPY-FL (used as standard). The amount of protein in the same samplewas measured using a standard Bradford assay using Pierce Protein Assaykit (Pierce, Rockford, Ill.).

FLAG Capture Assay

Biotinylation of FLAG Antibody

A 4.4 mg/mL stock of FLAG M2 monoclonal antibody (SIGMA Chemical, St.Louis, Mo.) is diluted with equal volume of 100 mM sodium bicarbonate(˜15 mM final antibody concentration). Subsequently, NHS-LC-Biotin(Pierce Chemical, Rockford, Ill.) is added from a 2 mM stock (in DMF) toa final 150 mM. The reaction is incubated for 2 hours on ice. Themixture is then clarified by centrifugation in a microcentrifuge (14,000R.P.M.) for 2.5 minutes. Unreacted labeling reagent is removed by gelfiltration chromatography.

Preparation of FLAG Antibody Coated ELISA Plates

NeutrAvidin™ biotin binding protein (Pierce Chemical, Rockford, Ill.) isdiluted to a final concentration of 50 mg/mL in 100 mM sodiumbicarbonate and used to coat Microlite(2+white opaque 96-well ELISAplates (Dynex Technologies, Chantilly, Va.). Plates are washed withTBS-T and coated using a solution of 5 mg/mL biotinylated FLAG M2antibody in TBS-T. Plates are washed with TBS-T and blocked inTranslation Dilution Buffer (TDB) [4.5% Teleostean Gelatin, 2% non-fatmilk powder, 10 mM EDTA, 0.1% Tween-20, 1.25 mg/mL pre-immune mouse IgG,2.5 mM d-biotin, in TBS, pH 7.5.].

Binding and Detection of Target Protein

Triple-epitope-tagged target proteins produced by in vitro translationusing rabbit reticulocyte extract are diluted 1/25-1/75 in TDB and addedto the antibody coated ELISA plates. Following capture of the targetprotein, plates are washed with TBS-T. Detection of c-myc is performedusing a polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.) followed by a peroxidase labeled secondary antibody, whereasdetection of the His₆ tag is achieved with a peroxidase labeled nickelchelate-based probe (India(His Probe-HRP, Pierce, Rockford, Ill.).Antibodies are diluted in TDB and the India(His Probe-HRP is diluted inTBS-T supplemented with 5 mg/mL pre-immune mouse IgG. In all cases,signal is generated using a chemiluminescent substrate system.

His-Tag Metal Affinity Capture ELISA Assay

Binding and Detection of Target Protein

Triple-epitope-tagged target proteins produced by in vitro translationusing rabbit reticulocyte extract are diluted 1/25-1/75 in 1% BSA/TBS-Tand added to nickel chelate coated ELISA plates (Pierce Chemical,Rockford, Ill.). Following capture of the target protein, plates arewashed with TBS-T and blocked with 1% BSA/TBS-T. Detection of epitopetags on the bound target protein is achieved using a monoclonal FLAG M2antibody (SIGMA Chemical, St. Louis, Mo.) or a polyclonal c-myc antibody(Santa Cruz Biotechnology, Santa Cruz, Calif.) in conjunction with theappropriate peroxidase labeled secondary antibody. Detection of biotinincorporated into the target protein via Biotin-lysyl-tRNA^(lys) isachieved using NeutrAvidin™ biotin binding protein conjugated toperoxidase (Pierce Chemical, Rockford, Ill.). The NeutrAvidin™ conjugateand all antibodies are diluted in 1% BSA/TBS-T. In all cases, signal isgenerated using a chemiluminescent substrate system.

Mass Spectrometry-Based Protease Assay

100 picomoles of the synthetic R6 peptide in 1 microliter of water isadded to 5 ul of a test solution and incubated for 20 minutes (or 10minutes) at 37 degrees. 50 ul of PBS is then added to the test mixtureand the resulting solution is passed over a 1 microliter anti-FLAGmicrocolumn (which recognizes the epitope sequence at the N terminus ofthe R6 peptide). The column is washed with 50 ul of deionized water andthe bound peptides are eluted directly onto the maldi plate using 1microliter of CHCA matrix in 50% acetonitrile/0.2% TFA/49.8% deionizedwater. The intact R6 peptide and degradation products are detected byMaldi-Tof mass spectrometry in positive ion linear mode.

Example 1 Protecting Groups

As discussed above, the present invention contemplatesprotease-sensitive peptides in order to monitor for complicatingprotease activity. In some embodiments, the peptide contains aprotecting group on the N-terminus, in order to restrict exodigestion tothe C-terminus. A variety of protecting groups can be used. In thisexample, Fmoc is attached to a modified amino acid. Coumarin amino acid(1.14 mmol) was reacted with FluorenylmethyloxycarbonylN-hydroxysuccininmidyl ester (Fmoc-NHS ester) 1.08 mmol) in the presenceof 1.14 mmol of triethylamine for 30 minutes at room temperature. Thereaction mixture was acidified and the precipitate washed with 1 N HCland dried.

Example 2 Preparation of Extract and Template

Preparation of extract: Wheat germ embryo extract was prepared byfloatation of wheat germs to enrich for embryos using a mixture ofcyclohexane and carbon tetrachloride (1:6), followed by drying overnight(about 14 hours). Floated wheat germ embryos (5 g) were ground in amortar with 5 grams of powdered glass to obtain a fine powder.Extraction medium (Buffer I: 10 mM trisacetate buffer, pH 7.6, 1 nMmagnesium acetate, 90 mM potassium acetate, and 1 mM DTT) was added tosmall portions until a smooth paste was obtained. The homogenatecontaining disrupted embryos and 25 ml of extraction medium wascentrifuged twice at 23,000×g. The extract was applied to a SephadexG-25 fine column and eluted in Buffer II (10 mM trisacetate buffer, pH7.6, 3 mM magnesium acetate, 50 mM potassium acetate, and 1 mM DTT). Abright yellow band migrating in void volume and was collected (S-23) asone ml fractions which were frozen in liquid nitrogen.

Preparation of template: Template DNA was prepared by linearizingplasmid pSP72-bop with EcoRI. Restricted linear template DNA waspurified by phenol extraction and DNA precipitation. Large scale mRNAsynthesis was carried out by in vitro transcription using theSP6-ribomax system (Promega; Madison, Wis.). Purified mRNA was denaturedat 67 C for 10 minutes immediately prior to use.

Example 3 Cell-Free Translation Reactions

The incorporation mixture (100 ul) contained 50 ul of S-23 extract, 5 mMmagnesium acetate, 5 mM Tris-acetate, pH 7.6, 20 mM Hepes-KOH buffer, pH7.5; 100 mM potassium acetate, 0.5 mM DTT, 0.375 mM GTP, 2.5 mM ATP, 10mM creatine phosphate, 60 ug/ml creatine kinase, and 100 ug/ml mRNAcontaining the genetic sequence which codes for bacterioopsin.Misaminoacylated PCB-lysine or coumarin amino acid-tRNA^(lys) moleculeswere added at 170 ug/ml and concentrations of magnesium ions and ATPwere optimized. The mixture was incubated at 25 C for one hour.

Example 4 Isolation of Nascent Proteins Containing PCB-Lysine

Streptavidin coated magnetic Dynabeads M-280 (Dynal; Oslo, Norway),having a binding capacity of 10 ug of biotinylated protein per mg ofbead. Beads at concentrations of 2 mg/ml, were washed at least 3 timesto remove stabilizing BSA. The translation mixture containing PCB-lysineincorporated into nascent protein was mixed with streptavidin coatedbeads and incubated at room temperature for 30 minutes. A magnetic fieldwas applied using a magnetic particle concentrator (MPC) (Dynal; Oslo,Norway) for 0.5-1.0 minute and the supernatant removed with pipettes.The reaction mixture was washed 3 times and the magnetic beads suspendedin 50 ul of water.

Photolysis was carried out in a quartz cuvette using a Black-Raylongwave UV lamp, Model B-100 (UV Products, Inc.; San Gabriel, Calif.).The emission peak intensity was approximately 1100 uW/cm² at 365 nm.Magnetic capture was repeated to remove the beads. Nascent proteinsobtained were quantitated and yields estimated at 70-95%.

Example 5 One Triple Marker Embodiment

In this example, a three marker system is employed to detect nascentproteins, i.e. an N-terminus marker, a C-terminus marker, and anaffinity marker (the latter being an endogenous affinity marker). Theexperiment involves 1) preparation of a tRNA with a marker, so that amarker can be introduced (during translation) at the N-terminus of theprotein; 2) translation of hemolysin with nucleic acid coding for wildtype and mutant hemolysin; and 4) quantitation of the markers.

1. Preparation of biotin-methionyl-tRNA^(fmet)

The purified tRNA^(fmet) (Sigma Chemicals, St. Louis, Mo.) was firstaminoacylated with methionine. The typical aminoacylation reactioncontained 1500 picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HClbuffer, pH 7.5, 10 mM MgCl₂, 1 mM methionine, 2 mM ATP, 150 mM NaCl andexcess of aminoacyl tRNA-synth-etases (Sigma). The reaction mixture wasincubated for 45 min at 37° C. After incubation, the reaction mixturewas neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 andsubjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5volumes) was added to the aqueous phase and the tRNA pellet obtained wasdissolved in the water (25 ul). The coupling of NHS-biotin to the -aminogroup of methionine was carried out in 50 mM sodium bicarbonate buffer,pH 8.0 by incubating the aminoacylated tRNA^(fmet) (25 ul) withNHS-biotin (final concentration=2 mM) for 10 min at 0° C. and thereaction was quenched by the addition of free lysine (finalconcentration=100 mM). The modified tRNA was precipitated with ethanoland passed through Sephadex G-25 gel filtration column (0.5×5 cm) toremove any free reagent, if present.

2. In vitro Translation of alpha-HL DNA

A WT and Amber (at position 135) mutant plasmid DNA was using coding foralpha-hemolysin (alpha-HL), a 32 kDa protein bearing amino acid sequenceHis-His-His-His-His-His (His-6) (SEQ ID NO: XXX) at its C-terminal. Invitro translation of WT and amber mutant alpha-HL gene (Amb 135) wascarried out using E. coli T7 circular transcription/translation system(Promega Corp., Wisconsin, Wis.) in presence ofBiotin-methionyl-tRNA^(fmet) (AmberGen, Inc.). The translation reactionof 100 ul contained 30 ul E. coli extract (Promega Corp., Wisconsin,Wis.), 40 ul premix without amino acids, 10 ul amino acid mixture (1mM), 5 ug of plasmid DNA coding for WT and mutant alpha-HL, 150picomoles of biotin-methionyl-tRNA^(fmet) and RNase-free water. Thepremix (1×) contains 57 mM HEPES, pH 8.2, 36 mM ammonium acetate, 210 mMpotassium glutamate, 1.7 mM DTT, 4% PEG 8000, 1.25 mM ATP, 0.8 mM GTP,0.8 mM UTP, 0.8 mM CTP, 60 mM phosphoenol pyruvate, 0.6 mM cAMP and 6 mMmagnesium acetate. From the translation reaction premix,n-formyl-tetrahydrofolate (fTHF) was omitted. The translation wascarried out at 37° C. for 1 hour. The translation reaction mixtureincubated without DNA is taken as control. After the translationreaction mixture was diluted with equal volume of TBS (Tris-bufferedsaline, pH 7.5). Each sample was divided into two aliquots and processedindividually as described below.

3. Preparation of Anti-Alpha-HL Antibody Microtiter Plate

Anti-rabbit-IgG coated microtiter plate (Pierce Chemicals, Rockford,Ill.) was washed with Superblock buffer solution (Pierce) and incubatedwith 100 ug/ml of anti-alpha-HL polyclonal antibody solution (SigmaChemicals, St. Louis, Mo.) prepared in Superblock buffer on microtiterplate shaker for 1 hour at room temperature. The plate was then washed(3 times×200 ul) with Superblock buffer and stored at 4° C. till furtheruse.

4. Quantitation of N-Terminal (Biotin) Marker

The translation reaction mixture (50 ul) for the control, WT and amberalpha-HL DNA were incubated in different wells of anti-alpha-HLmicrotiter plate for 30 minutes on the shaker at room temperature. Afterincubation, the wells were washed 5 times (5-10 min each) with 200 ulSuperblock buffer and the supernatant were discarded. To these wells,100 ul of 1:1000 diluted streptavidin-horse radish peroxidase(Streptavidin-HRP; 0.25 mg/ml; Promega) was added and the plate wasincubated at room temperature for 20 min under shaking conditions. Afterthe incubation, excess streptavidin-HRP was removed by extensive washingwith Superblock buffer (5 times×5 min each). Finally, 200 ul ofsubstrate for HRP (OPD in HRP buffer; Pierce) was added and the HRPactivity was determined using spectrophotometer by measuring absorbanceat 441 nm.

5. Quantitation of C-Terminal (His-6-taq) Marker

Translation reaction mixture (50 ull) from example 2 for control, WT andAmber alpha-HL DNA were incubated in different wells of anti-alpha-HLmicrotiter plate for 30 min on the shaker at room temperature. Afterincubation, the wells were washed 5 times (5-10 min each) with 200 ulSuperblock buffer and the supernatant were discarded. To these wells,100 ul of 1:1000 diluted anti-His-6 antibody (ClonTech, Palo Alto,Calif.) was added to the well and incubated at room temperature for 20min under shaking conditions. After the incubation, excess antibodieswere removed with extensive washing with Superblock buffer (5 times×5min each). Subsequently, the wells were incubated with secondaryantibody (anti-mouse IgG-HRP, Roche-BM, Indianapolis, Ind.) for 20 minat room temperature. After washing excess 2^(nd) antibodies, HRPactivity was determined as described above.

6. Gel-Free Quantitation of N- and C-Terminal Markers

The results of the above-described quantitation are shown in FIG. 5A(quantitation of N-terminal, Biotin marker) and FIG. 5B (quantitation ofC-terminal, His-6 marker). In case of in vitro transcription/translationof WT alpha-HL DNA in presence of biotin-methionyl-tRNA, the proteinsynthesized will have translated His-6 tag at the C-terminal of theprotein and some of the alpha-HL molecules will also carry biotin attheir N-terminus which has been incorporated usingbiotinylated-methionine-tRNA. When the total translation reactionmixture containing alpha-HL was incubated on anti-alpha-HL antibodyplate, selectively all the alpha-HL will bind to the plate viainteraction of the antibody with the endogenous affinity marker. Theunbound proteins can be washed away and the N- and C-terminal of thebound protein can be quantitated using Streptavidin-HRP and anti-His-6antibodies, respectively. In case of WT alpha-HL, the protein will carryboth the N-terminal (biotin) and C-terminal (His-6) tags and hence itwill produce HRP signal in both the cases where streptavidin-HRP andsecondary antibody-HRP conjugates against His-6 antibody used (HL, FIG.5A). On the other hand, in case of amber mutant alpha-HL, onlyN-terminal fragment of alpha-HL (first 134 amino acids) will be producedand will have only N-terminal marker, biotin, but will not have His-6marker due to amber mutation at codon number 135. As a result of thismutation, the protein produced using amber alpha-HL DNA will bind to theantibody plate but will only produce a signal in the case ofstrepavidin-HRP (HL-AMB, FIG. 5A) and not for anti-HisX6 antibodies(HL-AMB, FIG. 5B).

Example 6 Incorporation of Three Markers into Hemolysin

This is an example wherein a protein is generated in vitro underconditions where N- and C-terminal markers are incorporated along with amarker incorporated using a misaminoacylated tRNA. The Exampleinvolves 1) PCR with primers harboring N-terminal and C-terminaldetectable markers, 2) preparation of the tRNA, 3) in vitro translation,4) detection of nascent protein.

1. PCR of alpha-Hemolysin DNA

Plasmid DNA for alpha-hemolysin, pT7-WT-H6-HL, was amplified by PCRusing following primers. The forward primer (HL-5) was:5′-GAATTC-TAATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATGGAACAAAAATTAATCTCGGAAGAGGATTTGGCAGATTCTGATATTAATATTAAAACC-3′ (SEQ ID NO:136)and the reverse primer (HL-3) was: 5′-AGCTTCATTA-ATGATGGTGATGG-TGGTGAC3′ (SEQ ID NO:137). The underlined sequence in forward primer is T7promoter, the region in bold corresponds to ribosome binding site(Shine-Dalgamo's sequence), the bold and underlined sequences involvethe C-myc epitope and nucleotides shown in italics are the complimentaryregion of alpha-hemolysin sequence. In the reverse primer, theunderlined sequence corresponds to that of HisX6 epitope. The PCRreaction mixture of 100 ul contained 100 ng template DNA, 0.5 uM eachprimer, 1 mM MgCl₂, 50 ul of PCR master mix (Qiagen, Calif.) andnuclease free water (Sigma Chemicals, St. Louis, Mo.) water. The PCR wascarried out using Hybaid Omni-E thermocyler (Hybaid, Franklin, Mass.)fitted with hot-lid using following conditions: 95° C. for 2 min,followed by 35 cycles consisted of 95° C. for 1 min, 61° C. for 1 minand 72° C. for 2 min and the final extension at 72° C. for 7 min. ThePCR product was then purified using Qiagen PCR clean-up kit (Qiagen,Calif.). The purified PCR DNA was used in the translation reaction.

2. Preparation of BODIPY-FL-lysyl-tRNA^(lys)

The purified tRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was firstaminoacylated with lysine. The typical aminoacylation reaction contained1500 picomoles (˜1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HCl buffer, pH 7.5,10 mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl and excess of aminoacyltRNA-synthetases (Sigma Chemicals, St. Louis, Mo.). The reaction mixturewas incubated for 45 min at 37° C. After incubation, the reactionmixture was neutralized by adding 0.1 volume of 3 M sodium acetate, pH5.0 and subjected to chloroform:acid phenol extraction (1:1). Ethanol(2.5 volumes) was added to the aqueous phase and the tRNA pelletobtained was dissolved in water (35 ul). To this solution 5 ul of 0.5 MCAPS buffer, pH 10.5 was added (50 mM final conc.) followed by 10 ul of10 mM solution of BODIPY-FL-SSE. The mixture was incubated for 10 min at0° C. and the reaction was quenched by the addition of lysine (finalconcentration=100 mM). To the resulting solution 0.1 volume of 3 MNaOAc, pH=5.0 was added and the modified tRNA was precipitated with 3volumes of ethanol. Precipitate was dissolved in 50 ul of water andpurified on Sephadex G-25 gel filtration column (0.5×5 cm) to remove anyfree fluorescent reagent, if present. The modified tRNA was storedfrozen (−70° C.) in small aliquots in order to avoid free-thaws. Themodification extent of the aminoacylated-tRNA was assessed by acid-ureagel electrophoresis. Varshney et al., J. Biol. Chem. 266:24712-24718(1991).

3. Cell-Free Synthesis of Proteins in Eukaryotic (Wheat Germ)Translation Extracts.

The typical translation reaction mixture (20 ul) contained 10 ul of TnTwheat germ extract (Promega Corp., Wisconsin-Madison, Wis.), 0.8 ul ofTnT reaction buffer, 2 ul of amino acid mix (1 mM), 0.4 ul of T7 RNApolymerase, 30 picomoles of BODIPY-FL-lysyl-tRNA^(lys), 1-2 ug plasmidor PCR DNA (Example 1) and RNase-free water. The translation reactionwas allowed to proceed for 60 min at 30° C. and reaction mixture wascentrifuged for 5 min to remove insoluble material. The clarifiedextract was then precipitated with 5-volumes of acetone and theprecipitated proteins were collected by centrifugation. The pellet wasdissolved in 1× loading buffer and subjected to SDS-PAGE after boilingfor 5 min. SDS-PAGE was carried out according to Laemmli, Nature,227:680-685.

4. Detection of Nascent Protein

After the electrophoresis, gel was scanned using FluorImager 595(Molecular Dymanics, Sunnyvale, Calif.) equipped with argon laser asexcitation source. For visualization of BODIPY-FL labeled nascentprotein, we have used 488 nm as the excitation source as it is theclosest to its excitation maximum and for emission, we have used 530±30filter. The gel was scanned using PMT voltage 1000 volts and either 100or 200 micron pixel size.

The results are shown in FIG. 6. It can be seen from the Figure that onecan in vitro produce the protein from the PCR DNA containing desiredmarker(s) present. In the present case, the protein (alpha-hemolysin)has a C-myc epitope at N-terminal and HisX6 epitope at C-terminal. Inaddition, BODIPY-FL, a fluorescent reporter molecule is incorporatedinto the protein. Lane 1: alpha-Hemolysin plasmid DNA control; lane 2:no DNA control; lane 3: PCR alpha-hemolysin DNA and lane 4: hemolysinamber 135 DNA. The top (T) and bottom (B) bands in all the lane are fromthe non-specific binding of fluorescent tRNA to some proteins in wheatgerm extract and free fluorescent-tRNA present in the translationreaction, respectively.

Example 7 Primer Design

It is not intended that the present invention be limited to particularprimers. A variety of primers are contemplated for use in the presentinvention to ultimately incorporate markers in the nascent protein (asexplained above). The Example involves 1) PCR with primers harboringmarkers, 2) in vitro translation, and 3) detection of nascent protein.

For PCR the following primers were used: forward primer:5′GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAACAAAAATTAATATCGGAAGAGGATTTGAATGTTTCTCCATACAGGTCACGGGGA-3′ (SEQ ID NO:138). ReversePrimer: 5′-TTATTAATGATGGTGATGGTGGTG-TTCTGTAGGAATGGTATCTCGTTTTTC-3′ (SEQID NO:139) The underlined sequence in the forward primer is T7 promoter,the bold and underlined sequences involve the C-myc epitope andnucleotides shown in italics are the complimentary region ofalpha-hemolysin sequence. In the reverse primer, the bold sequencecorresponds to that of His-6 epitope and the underlined sequencecorresponds to the complimentary region of the alpha-hemolysin sequence.In a preferred embodiment, the reverse primer further comprises asequence which will generate a stop codon if there is a frameshift:TTT-ATT-TAT. An example of such a design for a reverse primer is asfollows:5′-TTATTA-ATGATGGTGATGGTGGTG-TTTATTTAT-TTCTGTAGGAATGGTATCTCGTTTTTC-3′(SEQ ID NO: 140) (wherein the underlined bolded section shows thissequence. A PCR reaction mixture of 100 ul can be used containing 100 ngtemplate DNA, 0.5 uM each primer, 1 mM MgCl₂, 50 ul of PCR master mix(Qiagen, Calif.) and nuclease free water (Sigma Chemicals, St. Louis,Mo.) water. The PCR was carried out using Hybaid Omni-E thermocyler(Hybaid, Franklin, Mass.) fitted with hot-lid using followingconditions: 95° C. for 2 min, followed by 35 cycles consisted of 95° C.for 1 min, 61° C. for 1 min and 72° C. for 2 min and the final extensiona 72° C. for 7 min. The PCR product can then be purified using QiagenPCR clean-up kit (Qiagen, Calif.). The purified PCR DNA can then be usedin a variety of translation reactions. Detection can be done asdescribed above.

Overall, the present invention contemplates a variety of primer designsbased on the particular epitopes desired (see Table 3 for a list ofillustrative epitopes). In general, the epitopes can be inserted as theN-terminus or C-terminus. In addition, they can be used to introduce anaffinity region (i.e. a region which will bind to antibody or otherligand) into the protein.

Example 8 Antibody Detection of Primer-Encoded Epitopes

This is an example wherein a protein is generated in vitro underconditions where affinity regions are incorporated in a protein andthereafter detected. The Example involves 1) PCR with primers containingsequences that encode epitopes, 2) preparation of the tRNA, 3) in vitrotranslation, 4) detection of nascent protein.

1. PCR with Primer-Encoded Epitopes

The total RNA from the human colon (Clontech, Palo Alto, Calif.) wassubjected to one-step RT-PCR reaction using ClonTech RT-PCR Kit. Theforward Primer, PTT-T7-P53, was5′-GGATCCTAATACGACTCACTATAGGGAGACCACCA-TGGGACACCACCATCACCATCACGGAGATTACAAAGATGACGATGACAAA-GAGGAGCCGCAGTCAGATCCTAGCGTCGA-3′(SEQ ID NO:141) and the reverse primer, Myc-P53-3′, was5′-ATTATTACAAATCCTCTTCCGAGATTAATT-TTTGTTCGTCTGAGTCAGGCCCTTCTGTCTTGAACATG-3′(SEQ ID NO:142). The underlined sequence in forward primer is T7promoter, the nucleotides shown in italics corresponds to that of His-6tag while the sequence in bold codes for FLAG-epitope and the rest ofprimer is the complementary region for P53 DNA. In the reverse primer,the underlined sequence corresponds to that of c-Myc epitope.

The RT-PCR/PCR reaction mixture of 50 ul contained lug total human colonRNA, 0.5 uM each primer, 43.5 ul of RT-PCR master mix (ClonTech) andnuclease free water (Sigma Chemicals, St. Louis, Mo.) water. TheRT-PCR/PCR was carried out in PTC-150 thermocyler (MJ Research, Waltham,Mass.) using following conditions: 50 C for 1 hour, 95 C for 5 minfollowed by 40 cycles consisted of 95 C for 45 sec, 60 C for 1 min and70 C for 2 min and the final extension at 70 C for 7 min. The PCRproduct was analyzed on 1% agarose gel and the PCR amplified DNA wasused in the translation reaction without any further purification. Theartificial C-terminal truncated mutant of P53 was prepared using theidentical procedure described above except the reverse primer,3′-P53-Mut, was 5′-CTCATTCAGCTCTCGGAACATC-TCGAAGCG-3′ (SEQ ID NO:143).

2. tRNA Labelling

Purified tRNA^(lys) (Sigma Chemicals, St. Louis, Mo.) was firstamino-acylated with lysine. The typical aminoacylation reaction (100 ul)contained 1500 picomoles (−1.0 OD₂₆₀) of tRNA, 20 mM imidazole-HClbuffer, pH 7.5, 10 mM MgCl₂, 1 mM lysine, 2 mM ATP, 150 mM NaCl andexcess of aminoacyl tRNA-synthetases (Sigma). The reaction mixture wasincubated for 45 min at 37° C. After incubation, the reaction mixturewas neutralized by adding 0.1 volume of 3 M sodium acetate, pH 5.0 andsubjected to chloroform:acid phenol extraction (1:1). Ethanol (2.5volumes) was added to the aqueous phase and the tRNA pellet obtained wasdissolved in the water (35 ul). To this solution 5 ul of 0.5M CAPSbuffer, pH 10.5 was added (final concentration of 50 mM) followed by 10ul of 10 mM solution of BODIPY-FL-SSE. The mixture was incubated for 10minutes at 0° C. and the reaction was quenched by the addition of freelysine (final concentration=100 mM). To the resulting solution 0.1volumes of 3 M NAOAc (pH=5.0) was added and the modified tRNA wasprecipitated with 3 volumes of ethanol. Precipitate was dissolved in 50ul of RNase-free water and passed through Sephadex G-25 gel filtrationcolumn (0.5×5 cm) to remove any free fluorescent reagent, if present.The modified tRNA was stored frozen (−70° C.) in small aliquots in orderto avoid freeze-thaws. The modification extent of the aminoacylated-tRNAwas assessed by acid-urea gel electrophoresis [Varshney, U., Lee, C. P.& RajBhandary, U. L., J. Biol. Chem. 266, 24712-24718 (1991)] or by HPLC[Anal. Biochem. 279:218-225 (2000)].

3. Translation

Translation of P53 DNA (see step 1, above) was carried out in rabbitreticulocyte translation extract in presence of fluorescent-tRNA (step2, above).

4. Detection

Once the translation was over, an aliquot (5 ul) was subjected toSDS-PAGE and the nascent proteins were visualized using FluorImager SI(Molecular Dynamics, Sunnyvale, Calif.). After visualization, the gelwas soaked in the transfer buffer (12 mM Tris, 100 mM glycine and 0.01%SDS, pH 8.5) for 10 min. Proteins from the gels were then transferred toPVDF membrane by standard western blotting protocol using Bio-Radsubmersion transfer unit for 1 hr. After the transfer, then membrane wasreversibly stained using Ferrozine/ferrous total protein stain for 1 minto check the quality of transfer and then the membrane was blocked usingamber blocking solution (4.5% v/v teleostean gelatin, 2% w/v non-fatmilk powder, 0.1% w/v Tween-20 in Tris-buffered saline, pH 7.5) for 2hours followed by overnight incubation (12-15 hours at 4° C. on constantspeed shaker) with appropriately diluted antibodies. For Flag detection,we have used 2000-fold diluted anti-Flag M2 Antibody (Sigma), for His-6detection, we have used 500-fold anti-His6 antibody (Santa-Cruz Biotech,Calif.) and for c-Myc detection, we have used 500-fold dilutedanti-C-Myc antibody (Santa-Cruz Biotech, Calif.).

After primary antibody incubation, the membrane was washed with TBST(Tris-buffered saline, pH 7.5 with 0.1% Tween-20) four times (10 mineach wash) and incubated with appropriately diluted secondary antibodies(10,000-fold diluted) for 1 hour at room temperature on constant speedshaker. The unbound secondary antibodies were washed with TBST (4washes/10 min each) and the blot was visualized using an ECL-Pluschemiluminescence detection system (Amersham-Pharmacia Biotech, N.J.).

The results are shown in FIGS. 7A and 7B. FIG. 7A shows the totalprotein stain of PVDF membranes following protein transfer from the gelfor three replicate blots containing a minus DNA negative control and aplus p53 DNA sample respectively. FIG. 7B shows the same blots (totalprotein staining is reversible) are probed with antibodies against thethree epitope tags using standard chemiluminescent Western blottingtechniques. Arrows indicate the position of p53.

Example 9 Gel-Free PTT for Cancer Genes

Although the replacement of radioactivity with fluorescent labelsrepresents an improvement in current PTT technology, it still relies onthe use of gels, which are not easily adaptable for high-throughputscreening applications. For this reason, this example demonstrates anon-gel approach based on the use of chemiluminescent detection. In thisapproach, a cancer-linked protein or polypeptide fragment from theprotein is expressed in vitro from the corresponding gene with differentdetection and binding tags incorporated at the N-terminal, C-terminaland between the two ends of the protein using a combination of speciallydesigned primers and tRNAs. The detection and binding tags provide ameans to quantitate the fraction of protein or protein fragment which istruncated while the tags located between the two ends of the protein canbe used to determine the region of truncation. For example, afull-length protein would contain both an N and C-terminal tag, whereasa truncated protein would contain only the N-terminal tag. The signalfrom a tag incorporated at random lysines between the two ends of theprotein (intrachain signal) would be reduced proportional to the size ofthe truncated fragment. It is important to also capture the protein witha marker located close to the N-terminus in order to avoid interferenceof chain truncations with binding.

In order to evaluate this method, we performed experiments on the APCand p53 genes containing either a WT sequence or truncating mutations.In both cases, a combination of primers and specially designed tRNAswere used to incorporate a series of markers into the target proteinsduring their in vitro synthesis in a rabbit reticulocyte system. Afterin vitro expression, the expressed protein was captured in 96-well ELISAplates using an affinity element bound to the plate. The relative amountof N-terminal, C-terminal and intrachain signal was then determinedusing separate chemiluminescent-based assays.

1. PCR of Cancer Genes

A. APC Segment 3

First, the genomic DNA (WT and isolated from cell lines harboring mutantAPC gene) was amplified by PCR using following primers. The forwardprimer, PTT-T7-APC3, was5′-GGATCCTAATACGACTCACTATAGGGAGACCACCATGCACCACCATCACCATCACGGAGGAGATTACAAAGATGACGATGACAAA-GTTTCTCCATACAGGTCACGGGGAGCCAAT-3′(SEQ ID NO:144) and the reverse primer, PTT-Myc-APC3, was5′-ATTATTACAAATCCTCTTCCGAGATTAA-TTTTTGTTCACTTCTGCCTTCTGTAGGAATGGTATCTCG-3′(SEQ ID NO:145). The underlined sequence in forward primer is T7promoter, nucleotides shown in italics corresponds to that of His-6 tagwhile the nucleotides sequence shown in the bold codes for FLAG-epitopeand the rest of the primer is the complementary region for APC segment 3DNA. In the reverse primer, the underlined sequence corresponds to thatof c-Myc epitope. The PCR\reaction mixture of 50 ul contained 200-500 ngtemplate DNA (either WT or mutant), 0.5 uM each primer and 25 ul of PCRmaster mix (Qiagen, Calif.) and nuclease free water (Sigma Chemicals,St. Louis, Mo.) water. The PCR was carried out using Hybaid Omni-Ethermocyler (Hybaid, Franklin, Mass.) fitted with hot-lid usingfollowing conditions: 95° C. for 3 min, followed by 40 cycles consistingof 95 C for 45 sec, 55 C for 1 min and 72 C for 2 min and the finalextension at 72 C for 7 min. The PCR product was analyzed on 1% agarosegel and the PCR amplified DNA was used in the translation reactionwithout any further purification.

B. P53

The p53 DNA was prepared as described above.

2. Preparation of the tRNA

The BODIPY-FL-lysyl-tRNA^(lys) was prepared as described above.Preparation of Biotin-lysyl-tRNA^(lys) and PC-Biotin-lysyl-tRNA^(lys)was achieved as follows. The purified tRNA^(lys) (Sigma Chemicals, St.Louis, Mo.) was first aminoacylated with lysine. The typicalaminoacylation reaction contained 1500 picomoles (−1.0 OD₂₆₀) of tRNA,20 mM imidazole-HCl buffer, pH 7.5, 10 mM MgCl₂, 1 mM lysine, 2 mM ATP,150 mM NaCl and excess of aminoacyl-tRNA-synthetases (Sigma Chemicals,St. Louis, Mo.). The reaction mixture was incubated for 45 min at 37 CAfter incubation, the reaction mixture was neutralized by adding 0.1volume of 3 M sodium acetate, pH 5.0 and subjected to chloroform:acidphenol extraction (1:1). Ethanol (2.5 volumes) was added to the aqueousphase and the tRNA pellet obtained was dissolved in water (35 ul). Tothis solution 5 ul of 0.5 M CAPS buffer, pH 10.5 was added (50 mM finalconc.) followed by 10 ul of 10 mM solution of either Biotin orphotocleavable-Biotin. The mixture was incubated for 10 min at OC andthe reaction was quenched by the addition of lysine (finalconcentration=100 mM). To the resulting solution 0.1 volume of 3 MNaOAc, pH=5.0 was added and the modified tRNA was precipitated with 3volumes of ethanol. Precipitate was dissolved in 50 ul of water andpurified on Sephadex G-25 gel filtration column (0.5×5 cm) to remove anyfree fluorescent reagent, if present. The modified tRNA was storedfrozen (−70 C) in small aliquots in order to avoid free-thaws. Themodification extent of the aminoacylated-tRNA was assessed by acid-ureagel electrophoresis (Varshney, U., Lee, C. P. & RajBhandary, U. L.,1991, J. Biol. Chem. 266, 2471224718).

3. Translation

The typical translation reaction mixture (20 ul) contained 16 ul of TNTrabbit reticulocyte extract for PCR DNA (Promega, Madison, Wis.), 1 ulof amino acid mix (1 mM), 1-2 ul of PCR DNA (see APC and p53 preparationdescribed above) and RNase-free water. For fluorescence detection, theBODIPY-FL-lysyl-tRNA^(lys) was included into the translation reactionmixture. The translation reaction was allowed to proceed for 60 min at30 C.

4. Detection

FIG. 8A shows the results of an initial experiment designed to detect achain truncation introduced into the p53 protein during RT-PCR In thiscase an N-terminal FLAG epitope was used for capture (see thedescription of the capture assay using 96-well ELISA plates at thebeginning of the EXPERIMENTAL section), and His₆ and c-myc used for theN- and C-terminal markers, respectively. Detection of the N-terminusHis-tag was achieved using a peroxidase labeled nickel chelate-baseddetection probe (India™ His Probe-HRP, Pierce, Rockford, Ill.).Detection of the C-terminus was performed using a rabbit polyclonalantibody directed against the human c-myc epitope followed by aperoxidase labeled mouse anti-[rabbit IgG] secondary antibody. As seen,the ratio of C/N terminal signals is reduced approximately 25-fold forthe truncated protein compared to WT. Further optimization of this assayshould result in sensitivity sufficient to detect truncating mutationsin 1/100 mutant/WT p53 proteins, thus enabling applications tonon-invasive colon cancer screening.

In a second experiment (FIG. 8B), capture was facilitated with anN-terminal His₆-tag, while FLAG and c-myc were used as N and C-terminalmarkers, respectively. In addition, an intrachain photocleavable biotinmarker was incorporated by adding PC-Biotin-Lys-tRNA to the in vitromixture. Biotin detection was achieved using peroxidase labeledNeutrAvidin™ (Pierce). The results show a 13-fold reduction in the C/Nratio for truncated p53 compared to WT. Furthermore, the intrachainbiotin signal drops by 75% relative to the N-terminal signal.

A third chemiluminescent protein truncation assay was designed to detectchain truncation in the APC gene of a mutant cell line (FIG. 8C).Capture was facilitated with an N-terminal His₆-tag, while FLAG andc-myc were used as N and C-terminal markers, respectively. As seen inFIG. 8C, the truncated APC exhibits a marked drop in the C/N ratio (1/6)again indicating the presence of a chain truncation.

Overall, these experiments demonstrate the ability to detectchain-truncating mutations in cancer-linked proteins using a gel-freechemiluminescent approach.

Example 10 Detection of Protease Activity in Extracts

Genomic DNA and RNA (WT and APC mutant) was isolated from establishedcell lines CaCo-2 (C1), HCT-8 (C2) and SW480 (C3) as well as frompatient blood samples using commercially available kits (Qiagen,Valencia, Calif.). PCR amplification of a selected region of the APCgene (APC segment 3) was carried out using 250-500 ng of genomic DNA,0.2 uM primer mix (forward and reverse) and 1×PCR master mix (Qiagen,Valencia, Calif.). Amplification was performed as follows: an initialcycle of denaturation at 95° C., forty cycles of denaturation at 95° C.for 45 sec, annealing at 57° C. for 45 sec, extension at 72° C. for 2min and a final extension step at 72° C. for 10 min. RT-PCRamplification of APC gene (APC segment 3) was carried out using one-stepRT-PCR/PCR kit from ClonTech (Palo Alto, Calif.). RT-PCR reactioncontained 500 ng of total RNA, 0.2 uM primer mix (forward and reverse)and 1× RT-PCR master mix. Amplification conditions were the same asabove with an additional initial cycle of reverse transcription at 50°C. for 1 hour. The primer pair was: Forward: (SEQ ID NO: 146)5′-GGATCCTAATACGACTCACTATAGGGAGACCACC-ATG-GGC-TACACCGACAT-CGAGATGAACCGCCTGGCAAG-GTTTCTCCATACAGGTCACGGGGAGCC-3′Reverse: (SEQ ID NO:147)5′-TTATTACAGCAGCTTGTGCAGGTCGCTGAAGGTACTTCTGCCTTCTGTAGGAATGTATC-3′

The italicized nucleotides in the forward primer correspond to the T7promoter, the underlined ATG is the initiation codon, the boldfacenucleotide region codes for the N-terminal tag (VSV-G; YTDIEMNRLGK: SEQID NO:148) and the remaining nucleotide sequences correspond to thecomplementary region of the APC gene. In the reverse primer, theboldface nucleotides code for the C-terminal tag (P53 sequence derivedtag; TFSDLHKLL: SEQ ID NO:149) while the rest of the nucleotide sequenceis complementary to the APC gene and nucleotides in italics codes for 2successive stop codons. After amplification, the quality and quantity ofthe PCR products was analyzed by agarose gel electrophoresis.

The cell-free reaction mixture contained 8 μl of TNT T7 Quick RabbitReticulocyte lysate for PCR DNA (Promega, Madison, Wis.), 0.5 μl of acomplete amino acid mix and 0.5 μl of DNA (approximately 200 ng) and 1μl of biotin-lysyl-tRNA. The translation reaction was allowed to proceedfor 30 min at 30° C. After the incubation, the reaction mixture wasdivided into two aliquots and to one, cycloheximide (1 uM) was added andfurther incubated up to 240 min. Aliquots (10 ul) were taken at 30, 60,120 and 240 min and the N-terminal and C-terminal signals weredetermined by ELISA as given below.

After the incubation of translation mixture, the reaction mixture wasdiluted 30-fold with TBS containing 0.05% Tween-20, 0.1% Triton X-100,5% BSA, and both antibodies (anti-VSV-G-HRP (Roche Applied Sciences,Indianapolis, Ind.) at 80 ng/mL and anti-p53-alkaline phosphatase (SantaCruz Biotechnology, Santa Cruz, Calif.) at 100 ng/mL). Subsequently, 100μl of the diluted reaction mixture was added to each well of aNeutrAvidin™ coated 96-well plate (pre-blocked with 5% BSA) andincubated for 45 min on an orbital shaker. NeutrAvidin™ was obtainedfrom Pierce Chemicals (Rockford, Ill.) and Microlite2+ multiwell plateswere obtained from Dynex Technologies (Chantilly, Va.). The plate waswashed 5× with TBS-T (TBS with 0.05% Tween-20) followed by 2× with TBSand developed using a chemiluminescent alkaline-phosphatase (AP)substrate (Roche Biochemicals, Indianapolis). After the AP readings,plates was washed with 2 times with TBS and HRP signal was measuredusing chemiluminescent HRP substrate (Supersignal Femto, PierceChemicals, Rockford, Ill.).

The results of effect of the addition of cycloheximide (CH) on thetranslation and N- and C-terminal signal are shown in the FIG. 9. It isclear from the data obtained that the addition of cycloheximide resultsin the rapid decrease in N- and C-terminal signals (+CH VSV signal and+CH p53 signal) since the protein synthesis is completely inhibited andthe nascent protein synthesized before the addition of cycloheximide isdegraded by proteases present in the translation extract. On the otherhand, when the cycloheximide was not added (i.e. protein synthesis wasnot inhibited), the decrease in N- and C-terminal signals is much slower(−CH VSV signal and −CH p53 signal) indicating the equilibrium processbetween synthesis and degradation. These results clearly indicate thepresence of protein degrading (proteolytic) activities in the rabbitreticulocyte extract.

Example 11 A Penta-Lysine 5′-Tag

The use of biotin-lysyl-tRNA to incorporate biotin affinity tags atlysine residues would result in no capture if the chain truncationoccurs upstream of the first lysine. This problem and the overallefficiency of capture can be improved if an extra lysine sequence areartificially added in the beginning the transcript. This can be achievedby adding 5 extra lysine coding nucleotides in the 5′ primer after anATG codon or after the epitope coding nucleotides. This design of thetranscript then can increase the overall number of lysine residuesresulting in the increased incorporation of biotin usingbiotin-tRNA^(lys). It is contemplated that the lysine tag includes, butis not limited to, from between 3-10 lysine residues.

FIG. 10 depicts ELISA data showing increased signal in samples havingextra lysine residues using shorter nascent proteins (WT is 70 kDnascent protein while is N3 is a mixture of 70 and 30 kD nascentproteins).

Example 12 MALDI-Mass Spectrometry (MALDI-MS) Mutation Detection

This example utilizes alpha-HL with a C-terminal His6-tag, which wasexpressed in a S30 reaction mixture using a high-expression plasmidcontaining the alpha-HL gene under control of the T7 promoter.

For comparison, a mutant of alpha-HL (S302W) was also expressed in E.coli translation extract. In both cases, the proteins were isolated fromthe translation reaction mixture using Co²⁺-NTA chromatography. Theisolated protein was dialyzed, concentrated and deposited on a MALDIsubstrate. As seen in FIG. 11, a peak is observed at 34,884 Da for WTalpha-HL and 34,982 Da for the mutant alpha-HL, in good agreement withcalculated masses (34,890 and 34,989 Da, respectively). Thisdemonstrates the ability of MALDI to detect a mutation in an in vitroexpressed protein.

Example 13 Detection of Protease Activity with Epitope Peptides

Two biotinylated peptides, a VSV peptide with an amino aid sequenceMYTDIEMNRLGK (SEQ ID NO:150) and P53 peptide with an amino acid sequenceTFSDLWKLL (SEQ ID NO:151) were synthesized. These peptides were used todetect the presence of proteolytic activities in the cell-freetranslation extracts such as rabbit reticulocyte, E. coli and Wheatgerm. In one experiment, 3 pmol of biotinylated VSV peptide wasincubated at 30° C. in either 100 ul Tris-buffer saline, rabbitreticulocyte extract or heat denatured rabbit reticulocyte extract (byboiling at 100° C. for 5 min in presence of SDS). In other experiment,10 pmol of biotinylated P53 peptide was incubated at 30° C. in either100 ul Tris-buffer saline, rabbit reticulocyte extract or heat denaturedrabbit reticulocyte extract (by boiling at 100° C. for 5 min in presenceof SDS). At a given time interval, 10 ul aliquots were removed andsubjected to ELISA assay as described above. The results of theseexperiments are presented in FIGS. 12 and 13. It can be seen from theFIG. 12 that when the VSV peptide was incubated in buffer only, no lossof peptide signal was observed. On the other hand, when the peptide wasincubated in rabbit reticulocyte lysate, complete loss of peptide signalwas observed within 10 min of incubation. Similar results were alsoobtained for p53 peptide (FIG. 13). When the peptide was incubated indenatured rabbit reticulocyte extract, it retains complete peptidesignal. These results show that there is significant protein/peptidedegrading activity present in the cell-free translation extract.

Example 14 Impact of Protease Inhibitors on Extract Protease Activity

The peptides (VSV and P53) were incubated with rabbit reticulocyteextract treated with protease inhibitor cocktail “PIC” (Roche AppliedSciences, Indianapolis, Ind.). After 30 min of protease cocktailtreatment, these peptides were incubated in the treated translationextract for 5-45 minutes and at given time interval 10 ul aliquots wereremoved. The residual peptide signal was then determined by ELISA asdescribed before. The results of this experiment are presented in FIGS.14 and 15. It can be seen from the FIG. 14 that when the peptide wasincubated in buffer alone, no loss of peptide signal was observed. Onthe other hand, when the peptide was incubated in rabbit reticulocytelysate, complete loss of peptide signal was observed within 10 min ofincubation. This complete loss of VSV peptide signal can be avoided bypre-treating the rabbit reticulocyte extract with a protease inhibitorcocktail. The P53 peptide required higher amounts of protease cocktailinhibitor (FIG. 15) and was subject to increased degradation over time(even at the higher inhibitor concentration). These results show thatthere is significant protein/peptide degrading activity present in thecell-free translation extract and this proteolytic activity can bepartially inhibited by pre-treating the translation extract with aprotease inhibitor cocktail.

Example 15 Protease Inhibitors and Nascent Protein Production

Even if protease inhibitors can reduce degradation from exposure to theextract, there is the concern that such inhibitors will interfere withprotein production. PCR product from example 10 was used for theproduction of nascent protein using rabbit reticulocyte lysate in thepresence of various amounts of protease inhibitor cocktail (RocheApplied Sciences, Indianapolis, Ind.). The translation was carried outas described. The translation was allowed to proceed for 45 min and theamount of nascent protein produced was determined by the ELISA assay.Briefly, the biotinylated nascent protein was captured on theneutravidin coated plate and the amount of nascent protein produced wasdetermined by measuring N-terminal (VSV) and C-terminal (P53) signalusing anti-VSV-HRP and anti-P53-AP antibody, respectively. The resultsof the translation reaction in the absence and presence of proteaseinhibitor are shown in the FIG. 16 (1× refers to the concentrationsuggested by the vendor). It can be seen from the Figure that thetranslation efficiency is not significantly inhibited up to the 5×concentration of the inhibitor mix. However, at higher concentrationsthere is a significant reduction.

Example 16 Protease Activity in a Reconstituted System

As shown above, the various commercially available translation extractssuch as rabbit reticulocyte and E. coli extract (Promega Corp., Madison,Wis.) contain significant protease activity. In this experiment, theprotease activity of a newly available reconstituted system (the “PURE”translation system) is evaluated (Post genome Institute, Japan). In thisexperiment, 1 pmol of biotinylated VSV peptide was incubated at 30° C.in either 100 μl Tris-buffer saline, rabbit reticulocyte extract or heatdenatured rabbit reticulocyte extract (by boiling at 100° C. for 5 minin presence of SDS), E. coli or heat denatured E. coli translationextracts as well as PURE or denatured PURE translation extract. At agiven time interval (5 and 45 min), 10 ul aliquots were removed andsubjected to ELISA assay as described above. The results of thisexperiment are presented in FIG. 17. It can be seen from the Figure thatwhen the peptide was incubated in a buffer alone, no loss of peptidesignal was observed. On the other hand, when the peptide was eitherincubated in rabbit reticulocyte lysate or E. coli lysate, complete lossof peptide signal was observed within 5 min of incubation. In addition,PURE system, which is made of purified translation machinery components,shows significant proteolysis for the P53 peptide at 5 min and increasedpeptide degradation when the incubation was prolonged to 45 min (eventhough the “PURE” system is advertised to be protease-free). Again, whenthe peptide was incubated with heat-denatured extracts, it retainedcomplete peptide signal. These results show that even reconstitutedsystems contain protease activity.

Example 17 Detection of Protease Activity by Mass Spectrometry

In this experiment, the protease-sensitive peptide (“R6”) was employed,which has a sequence of MDYKDDDDKRRRRRRFFF (SEQ ID NO:152). The residuesin italics correspond to the FLAG epitope located at the N-terminus. Thepeptide concentration used was either 1 nmole/μL or 100 pmole/μL. In oneexperiment, 10 μL of RRL extract was mixed with either 2 μL (2 nmoletotal peptide amount) or 5 μL (50 pmole) peptide solution and incubatedfor 30 s, 2 min or 5 min at 30° C. The reaction was terminated byaddition of 100 μL of 100 mM EDTA and the solution was immediatelyapplied to the microcolumn containing 1 μL of Sigma ANTI-FLAG beads. Thebeads were then washed with 50 μL of 20 mM Tris-HCl, pH 7.2 and thebound peptide was eluted with approximately 4 μL of matrix solution(CHCA/acetonitrile/TFA) directly onto a MALDI plate. In a controlexperiment, 5 μL (50 pmole) of pure peptide solution was applied to themicrocolumn containing ANTI-FLAG beads, washed and eluted as describedabove.

Incubation of 50 pmole of peptide with the buffer shows a very good masspeak at 2534 (FIG. 18, top panel). The peak at 1268 is doubly chargedspecies of the same peptide. Incubating this peptide in rabbitreticulocyte lysate even for 1 min results in a partial disappearance ofthe peak corresponding to the intact peptide, which is observed near2535 in the mass-spectra as well as the appearance of several peptidesof smaller masses (FIG. 18, middle panel) which can be identified as theproducts of exo-proteolysis. In particular, weak bands near 2387 and2238 are close to the expected peptide fragments, which lack one and twophenylalanines, respectively. The peak corresponding to the peptidefragment with all three Phe groups removed is either very weak orabsent. Peptide fragments with three Phe groups and 1, 2, 3 and 4 Arggroups removed give rise to peaks observed at 1934, 1778, 1621 and 1268,respectively. The peak at 1268 is due to the intact peptide (M²⁺).Furthermore, incubation of this peptide in rabbit reticulocyte extractfor 5 min results in complete loss of intact peptide (FIG. 18, bottompanel). Since the purification procedure requires the intact FLAGepitope to be present, it was not possible to detect fragments thatcorrespond to N-terminal degradation (if such fragments exist). Theresults presented here demonstrate that the RRL system is capable ofrapidly degrading picomolar amounts of short peptides. The observedpeaks indicate that the proteolysis occurs from the C-terminal end andtherefore the protease might be a carboxypeptidase.

Example 18 Inhibiting Protease Activity Detected by Mass Spectrometry

In this experiment, protease inhibitors were tested to inhibit thedegradation of the peptide R6 (described above). In one experiment, 10μL of rabbit reticulocyte extract was pre-treated by incubating with aspecific protease inhibitor such as ebelactone or 2-LeuLeuNVa-CHO for10-15 min. Following pretreatment, peptide R6 was added to abovetranslation mix and incubated for 10 min at 30° C. The reaction wasterminated by addition of 100 μL of 100 mM EDTA and the solution wasimmediately applied to the microcolumn containing 1 μL of SigmaANTI-FLAG beads. The beads were then washed with 50 μL of 20 mMTris-HCl, pH 7.2 and the bound peptide was eluted with approximately 4μL of matrix solution (CHCA/acetonitrile/TFA) directly onto a MALDIplate. In the control experiment, extract pre-treated with buffer wasused and processed as described above. Similar experiments were carriedout using E. coli translation extract.

Incubation of 50 pmole of peptide with the rabbit reticulocyte extracttreated with buffer (control) shows the disappearance of peptide peak(mass=2535) as a result of proteolysis (FIG. 19, Top panel). On theother hand, peptide incubated in rabbit reticulocyte extract which waspretreated with protease inhibitor (2-LeuLeuNvaCHO) shows a significantreduction in protease activity, which is evident from the detection of asignificant peptide peak appearing at its original mass corresponding toan intact R6 peptide (FIG. 19, Bottom panel). Another proteaseinhibitor, namely, Ebelactone, shows only weak protease inhibition (FIG.I, Middle panel). FIG. 20 (upper panel) shows that the commerciallyavailable E. coli translation extract contains significant proteaseactivity. Such activity in only weakly inhibited by Chymostatin (FIG.20, Bottom). On the other hand, significant inhibition is achieved withaprotinin (FIG. 20, middle). The results presented here clearlydemonstrate that both the RRL system and the E. coli system haveprotease at a level that is capable of rapidly degrading picomolaramounts of short peptides so as to complicate mass spec detection. Suchproteolysis activities present in the translation extracts can besignificantly inhibited by pre-treating translation reaction mixtureswith particular protease inhibitors.

Example 19 Inhibiting Proteolysis in Reconstituted Systems

All conventional translation systems tested (rabbit reticulocyte lysate,wheat germ extract, E. coli S30 extract) exhibited strong proteolyticactivity that results in a complete degradation of sub-nanomolar amountsof R6 peptide within several minutes after incubation with thetranslation mixtures. On the other hand, the reconstituted system(“PURE”) demonstrates a more specific proteolysis, leading to theaccumulation of a dominant truncated product (MDYKDDDDKRRRRR) (SEQ IDNO:153). In this example, inhibition of proteolysis in the reconstitutedsystems (PURE I and II) is explored in more depth.

A number of protease inhibitor products are available commerciallyincluding Complete™ tablets (Roche), Protease Inhibitor Cocktail (Sigma)and BioStab general proteolysis inhibitor (Fluka). These are mixtures ofindividual compounds with known inhibitory activity against serine,cysteine and metalloproteases. For the BioStab inhibitor the compositionand mechanism of inhibition are not described. The intended applicationfor these products is prevention of protein degradation duringpurification from the whole cell lysates. Neither these products nor theindividual protease inhibitors have been previously described assuitable for application in the in-vitro translation systems. There area number of reasons for this. First, the proteolytic activity inin-vitro translation systems, especially in the purified systems, suchas PURE, is expected to be significantly different from those in thewhole cell lysates. Second, and more important, is that some of theindividual components in the currently available protease inhibitormixtures also inhibit the protein synthesis. For example, EDTA acts asan ion chelator and causes depletion of Mg²⁺, which is required for thetranscription. Furthermore, protease inhibitor cocktails often requireDMSO as a solvent, which is not compatible with thetranscription/translation reactions. Therefore, the objective of thiswork was to find a set of compounds that prevent proteolytic degradationof in-vitro produced polypeptides but do not interfere with thetranscription and translation mechanisms. The R6 peptide was chosenbecause it possesses recognition sites for several types of proteasesincluding very common trypsin and chymotrypsin.

The proteolysis in reconstituted E. coli translation systems (PURE I andPURE II, Post-Genome Institute, Japan) was assayed as follows: 10 μL ofthe translation reaction containing all components except the DNA wasmixed with 1 μL of the R6 solution (10 pmol/μL) and incubated for 15 minat 37° C. 100 μL of the wash solution (1×PBS, 250 mM EDTA, 0.1% Triton)was added and applied to a microcolumn loaded with 1 μL of the anti-FLAGagarose beads (Sigma). The solution was allowed to pass through thebeads (approx. 5 min) and the beads were washed with the wash solutionand then 100 μL of dI H₂O. The peptide was eluted with the MALDI matrix(10 mg/mL α-hydroxycinnamic acid, 70% acetonitrile, 0.3% trifluoroaceticacid) solution directly onto a MALDI plate and analyzed bymass-spectrometry. To inhibit proteolysis in the reconstituted system,the assay was similar except that 1 μL of a protease inhibitor solutionwas added to the translation mixture and incubated for 15-30 min priorto the addition of the R6 peptide. More than 30 individual proteaseinhibitors (as well as combinations) were tested, including thefollowing compounds, which are inhibitors of serine, cysteine, acid andmetalloproteases as well as broad range inhibitors: α-macroglobulin,ebelactone B, bestatin, 6-aminohexanoic acid, phosphoramidon, EDTA,E-64, aprotinin, α-BOC deacetylleupeptin, AEBSF (Pefabloc™), ecotin,pepstatin, leupeptin, antipain, chymostatin, benzamidine HCl.

FIG. 21 (top trace) shows the disappearance of the intact R6 peptidewith the molecular weight of 2522 Da and appearance of the truncatedproduct (MDYKDDDDKRRRRR) (SEQ ID NO:153) with the molecular weight of1925 Da after 15 min incubation with PURE I mixture. On the other hand,the inhibitor AEBSF showed excellent inhibition. FIG. 22 shows verysimilar effect observed in the PURE II system with (bottom) and without(top) the AEBSF inhibitor. The vast majority of compounds tested in thePure II system were not effective, with the exception of AEBSF andaprotinin (FIG. 23). However, aprotinin was subsequently determined toinhibit protein synthesis. AEBSF could also inhibit protein synthesis ifused (as the manufacturer suggests) together with “Pefabloc protector.”Therefore, in the experiments described in this example, this“protector” was not used. Unlike the reconstituted E. coli resultsdescribed above, inhibiting the proteases in RR extracts could not bedone with a single compound to a degree necessary for mass spec analysis(see FIGS. 24 and 25). Various combinations were tested and severalpromising ones have been identified. In the data shown in FIG. 26 (lowertrace), the combination included antipain (stock concentration 20mg/mL), aprotinin (1 mg/mL), calpastatin (1 mg/mL) and α-BOCdeacetylleupeptin (5 mg/mL). 2 μL of the above protease inhibitorsolution was added to 10 μL of the translation mixture. Othercombinations were tested (FIG. 27) and they were either less effective(compare top panel to bottom panel) or completely ineffective (middlethree panels).

Example 20 Removing Proteases from Reconstituted Systems

Another approach to the protease problem is to remove proteases fromtranslation systems. The PURE kit purchased from the Post-GenomeInstitute contains two components: Solution A and Solution B, thecontents of which are not disclosed. The combined contents of these 2components comprise all elements needed to do in vitro transcription andtranslation from a PCR product including ribosomes, translation factors,an energy source, T7 polymerase, and ribonucleotides. When 1 ul ofsolutions A and B are analyzed by 1% agarose gel electrophoresis andstained with ethidium bromide, solution B shows a staining pattern thatresembles that obtained from ribosomes, while solution A does not.Analysis of solutions A and B using the mass spec-based protease assay(with R6 as described above) demonstrate that solution A has no proteaseactivity, while solution B has a protease that completely degrades theR6 test peptide by removing 4 residues from the C terminus. Thus,solution B contains ribosomes, contaminating proteases and perhaps otheressential factors required for in vitro translation.

In order to remove the protease activity from solution B, 100 ul ofsolution B was added to 200 ul of deionized water and spun through aYM100 Microcon filter with a 100 kDa cut-off (Millipore, Inc) at 10,000rpm for 5 minutes in a benchtop microfuge at room temperature. The R6mass spec-based protease assay showed that the filtrate greatly reducedprotease activity. However, when 3 ul of solution B filtrate was addedto 7 ul of solution A, the resulting mixture was unable to generate apeptide from a PCR product template. By contrast, when 10 picomoles ofzonally purified E. coli ribosomes (shown to be devoid of proteaseactivity using the R6 assay) in 1 ul of water are added to this mixture,translation does take place. 10 picomoles of zonally purified E. coliribosomes added to solution A without any solution B filtrate does notallow translation. Thus, solution B contains ribosomes but the filtratedoes not, while the filtrate does contain other essential factors fortranslation. The new translation mixture (hence forth referred to asAmber-PURE) can be dispensed in 10 ul aliquots and frozen at −70 degreesfor later use. A 10 ul aliquot contains 3 ul of diluted and filteredsolution B, 7 ul of solution A and 10 picomoles of zonally purifiedribosomes.

Example 21 Using Reconstituted Systems Depleted of Proteases

The previous example illustrates that one approach to the proteaseproblem of reconstituted systems is to remove proteases from translationsystems. In this example, the protease-depleted system is used toproduce the reference peptide (R6) by in vitro translation using DNAcoding for the R6 peptide. Template for the reaction consists of a PCRproduct encoding the R6 peptide that is made by filling in overlappingoligonucleotides of the following sequence:

Forward: (SEQ ID NO: 154)TAATACGACTCACTATAGGGAGGAGGACAGCTATGGACTACAAGGACGACGATGACAAGAGGAGGAGGAGGAGGAGGT Reverse: (SEQ ID NO: 155)CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTCAGAAGAAGAACCTCCTCCTCCTCCTCCTCTT

Addition of 1 ul of the PCR product to either 10 ul of PURE or 10 ul ofAmber-PURE followed by incubation at 37 degrees for 20 minutes resultsin the generation of the R6 test peptide (the predicted mass offormylated R6 is 2,553 Da). Most of the peptide products obtained fromPURE are smaller than the parent peptide and correspond to degradationproducts (FIG. 28A). This shows that the commercially availablereconstituted system as it is sold is not suitable for in vitrotranslation of polypeptides that are detected using a sensitive assaysuch as the mass spec-based assay described herein. In contrast, most ofthe peptide product obtained from the Amber-PURE is the intact patentpeptide and not degradation products (FIG. 28B). Thus, aprotease-depleted reconstituted system can be used for in vitrotranslation of polypeptides where detection is by mass spec.

Amber-PURE can also be used to produce peptides for genetic testingpurposes. For example, using the Amber-PURE system, the mass spectra ofa peptide encoded by a 210 base pair test sequence of the APC gene showsthe wild type peptide as well as mutants. More specifically, codons1299-1368 (70 codons containing 210 bases) of the APC gene wereamplified with the following primers:

Forward: (SEQ ID NO: 156)TAATACGACTCACTATAGGGAGGAGGACAGCTATGGACTACAAGGACGACGATGACAAGACGACACAGGAAGCAGATTCT Reverse: (SEQ ID NO: 157)TTTTTATGCGTAGTCTGGTACGTCGTATGGGTAGTGTTCAGGTGGACTTT TGGG

The forward primer contains a T7 polymerase binding sequence andsequence encoding the FLAG epitope that is used for purificationpurposes. The reverse primer encodes an HA epitope tag and provides astop codon. An additional reverse primer was used to encode a peptidehaving a His-Gly amino acid substitution:

Reverse His > Gly: (SEQ ID NO: 158)TTTTTATGCGTAGTCTGGTACGTCGTATGGGTAGCCTTCAGGTGGACTTT TGGG

These primers were used to amplify genomic DNA having the wildtype APCgene, or genomic DNA taken from tumor cell lines having truncatingmutations in the APC gene. The peptides encoded by the PCR products havethe following sequences and masses:

Wildtype (Mass 11,037): (SEQ ID NO: 159)MDYKDDDDKTTQEADSANTLQIAEIKEKIGTRSAEDPVSEVPAVSQHPRTKSSRLQGSSLSSESARHKAVEFSSGAKSPSKSGAQTPKSPPEHYPYDVPD YAMutant codon 1309 Del 5 (Mass 3,404 with formyl group at N terminus):

MDYKDDDDKTTQEADSANTLQIAEIKDWN (SEQ ID NO: 160)Mutant codon 1338 CAG>TAG (Mass 6,134 with formyl group at N terminus):

(SEQ ID NO: 161) MDYKDDDDKTTQEADSANTLQIAEIKEKIGTRSAEDPVSEVPAVSQHPRTKSSRLMutant codon 1367 CAG>TAG (Mass 8,981 with formyl group at N terminus):

(SEQ ID NO: 162) MDYKDDDDKTTQEADSANTLQIAEIKEKIGTRSAEDPVSEVPAVSQHPRTKSSRLQGSSLSSESARHKAVEFSSGAKSPSKSGAPoint mutation His(1374)>Gly(1374) (WT Mass 10,987 with formyl group atN terminus):

(SEQ ID NO: 163) MDYKDDDDKTTQEADSANTLQIAEIKEKIGTRSAEDPVSEVPAVSQHPRTKSSRLQGSSLSSESARHKAVEFSSGAKSPSKSGAQTPKSPPEGYPYDVPD YA

1 microliter of PCR product was added to 10 ul of Amber-PURE andincubated 20 minutes at 37 degrees. Synthesis was stopped by addition of100 ul of a solution containing 1% Triton-X 100 and 100 mM ammoniumbicarbonate(Tx100/ABC). This solution was then purified on a microcolumncontaining 1 ul of M2 anti-FLAG antibody coated sepharose beads andwashed with 100 ul Tx100/ABC and 100 ul distilled water. The boundpeptides were released with 1 ul of maldi matrix (10 mg/ml sinapinicacid, 0.1% TFA, 50% acetonitrile) directly onto a maldi plate andanalyzed with a Voyager DE-Pro Maldi-Tof mass spectrometer. The results(data not shows) reveal the dominant peak for each spectrum has theexpected mass of the encoded peptide; mutant peaks are easilydistinguished from the wildtype signal (even when the wild-type tomutant PCR template ratio is 4 to 1).

Example 22 Depleting Wild-Type Polypeptides

Detection of a mutant sequence is often complicated by the presence oflarge amounts of the wild-type sequence. For example, human APC genesisolated from stool samples will contain mostly the wild-type sequenceeven if the patient has a polyp containing a mutant version of the APCgene. Mass spectra of APC peptides made in vitro from stool sample DNAfrom such a patient will show a dominant wild-type signal that can makedetection of the truncated species difficult. Since the wild-typepeptide has an intact C-terminus and the mutant does not, it is possibleto deplete the wildtype peptides using an antibody against theC-terminal epitope (e.g. the HA epitope) present in the C-terminus.

This approach has been documented for a region of the APC genecontaining codons 1299-1317. The mutant truncated peptide was made froma sequence having the codon 1309 Del mutation (see Example 21, above).The formylated wildtype peptide has a mass of 5,769 Da, while the mutanttruncated peptide has mass of 3,404 Da. When the wild-type sequence(FIG. 29A) or the truncated peptide (FIG. 29B) are made alone, the peaksare quite evident. However, when the two are made together and thetemplate ratio is 1:256 (mutant:wild-type), the truncated peak is notreadily detected (FIG. 29C). On the other hand, if the peptide mixture,after peptide synthesis, is run over an anti-HA microcolumn containing 5ul of anti-HA agarose beads (Sigma, St Louis, Mo.), thereby depletingwild-type polypeptide sequences by binding to the C-terminal epitope,the truncated sequence is readily detected (FIG. 29D) even when startingwith a template ratio of 1:256 (mutant:wild-type). The results showdetection is readily achieved under conditions where the depletionprocess is incomplete and there is considerable wild-type sequenceremaining (indeed, from the peak heights one can estimate that thewild-type polypeptides are present in a ratio of at least 10:1 vis-à-visthe truncated peptide). Of course, more complete depletion protocols(e.g. optimizing affinity chromatography conditions by a) using a largercolumn, b) utilizing a higher affinity ligand:epitope system, or c)adjusting flow rates, etc.) can be employed to further enhancesensitivity.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered exemplary only, with the scope of particularembodiments of the invention indicated by the following claims.

TABLE 1 Truncation Mutations In Human Molecular Genetics DiseaseReferences % Truncating Mutations Gene Familial Adenomatous 95% APCPolyposis Hereditary desmold disease 100%  APC Ataxia telangiectasia 90%ATM Hereditary Breast and 90% BRCA1 Ovarian Cancer 90% BRCA2 CysticFibrosis 15% CFTR Duchenne Muscular 95% DMD Dystrophy Emery-DreifussMuscular 80% EMD Dystrophy Fanconi anaemia 80% FAA Hunter Syndrome 50%IDS Hereditary non-polyposis −80%  hMSH2 colorectal cancer 70% hMLH1Neurofibromatosis type 1 50% NF1 Neurofibromatosis type 2 65% NF2Polycystic Kidney Disease 95% PKD1 Rubinstein-Taybi Syndrome 10% RTS Thepercentage of truncating mutations reported which should be detectableusing PTT.

TABLE 2 PROTEASE-SENSITIVE PEPTIDES (n = 1 to 9) DYKDDDDKR_(n) (SEQ IDNO: 1) MDYKDDDDKR_(n) (SEQ ID NO: 7) DYKDDDDKR_(n)FFF (SEQ ID NO: 2)MDYKDDDDKR_(n)FFF (SEQ ID NO: 8) DYKDDDDKR_(n)YYY (SEQ ID NO: 3)MDYKDDDDKR_(n)YYY (SEQ ID NO: 9) DYKDDDDKR_(n)LLL (SEQ ID NO: 4)MDYKDDDDKR_(n)LLL (SEQ ID NO: 10) DYKDDDDKR_(n)DDD (SEQ ID NO: 5)MDYKDDDDKR_(n)DDD (SEQ ID NO: 11) DYKDDDDKR_(n)KKK (SEQ ID NO: 6)MDYKDDDDKR_(n)KKK (SEQ ID NO: 12) EQKLISEEDLR_(n) (SEQ ID NO: 13)MEQKLISEEDLR_(n) (SEQ ID NO: 19) EQKLISEEDLR_(n)FFF (SEQ ID NO: 14)MEQKLISEEDLR_(n)FFF (SEQ ID NO: 20) EQKLISEEDLR_(n)YYY (SEQ ID NO: 15)MEQKLISEEDLR_(n)YYY (SEQ ID NO: 21) EQKLISEEDLR_(n)LLL (SEQ ID NO: 16)MEQKLISEEDLR_(n)LLL (SEQ ID NO: 22) EQKLISEEDLR_(n)DDD (SEQ ID NO: 17)MEQKLISEEDLR_(n)DDD (SEQ ID NO: 23) EQKLISEEDLR_(n)KKK (SEQ ID NO: 18)MEQKLISEEDLR_(n)KKK (SEQ ID NO: 24) WSHPQFEKR_(n) (SEQ ID NO: 25)MWSHPQFEKR_(n) (SEQ ID NO: 31) WSHPQFEKR_(n)FFF (SEQ ID NO: 26)MWSHPQFEKR_(n)FFF (SEQ ID NO: 32) WSHPQFEKR_(n)YYY (SEQ ID NO: 27)MWSHPQFEKR_(n)YYY (SEQ ID NO: 33) WSHPQFEKR_(n)LLL (SEQ ID NO: 28)MWSHPQFEKR_(n)LLL (SEQ ID NO: 34) WSHPQFEKR_(n)DDD (SEQ ID NO: 29)MWSHPQFEKR_(n)DDD (SEQ ID NO: 35) WSHPQFEKR_(n)KKK (SEQ ID NO: 30)MWSHPQFEKR_(n)KKK (SEQ ID NO: 36) CSPFEVQVSPEAGAQKR_(n) (SEQ ID NO: 37)MCSPFEVQVSPEAGAQKR_(n) (SEQ ID NO: 43) CSPFEVQVSPEAGAQKR_(n)FFF (SEQ IDNO: 38) MCSPFEVQVSPEAGAQKR_(n)FFF (SEQ ID NO: 44)CSPFEVQVSPEAGAQKR_(n)YYY (SEQ ID NO: 39) MCSPFEVQVSPEAGAQKR_(n)YYY (SEQID NO: 45) CSPFEVQVSPEAGAQKR_(n)LLL (SEQ ID NO: 40)MCSPFEVQVSPEAGAQKR_(n)LLL (SEQ ID NO: 46) CSPFEVQVSPEAGAQKR_(n)DDD (SEQID NO: 41) MCSPFEVQVSPEAGAQKR_(n)DDD (SEQ ID NO: 47)CSPFEVQVSPEAGAQKR_(n)KKK (SEQ ID NO: 42) MCSPFEVQVSPEAGAQKR_(n)KKK (SEQID NO: 48) MASMTGGQQMGR_(n) (SEQ ID NO: 49) MMASMTGGQQMGR_(n) (SEQ IDNO: 55) MASMTGGQQMGR_(n)FFF (SEQ ID NO: 50) MMASMTGGQQMGR_(n)FFF (SEQ IDNO: 56) MASMTGGQQMGR_(n)YYY (SEQ ID NO: 51) MMASMTGGQQMGR_(n)YYY (SEQ IDNO: 57) MASMTGGQQMGR_(n)LLL (SEQ ID NO: 52) MMASMTGGQQMGR_(n)LLL (SEQ IDNO: 58) MASMTGGQQMGR_(n)DDD (SEQ ID NO: 53) MMASMTGGQQMGR_(n)DDD (SEQ IDNO: 59) MASMTGGQQMGR_(n)KKK (SEQ ID NO: 54) MMASMTGGQQMGR_(n)KKK (SEQ IDNO: 60) YPYDVPDYAR_(n) (SEQ ID NO: 61) MYPYDVPDYAR_(n) (SEQ ID NO: 67)YPYDVPDYAR_(n)FFF (SEQ ID NO: 62) MYPYDVPDYAR_(n)FFF (SEQ ID NO: 68)YPYDVPDYAR_(n)YYY (SEQ ID NO: 63) MYPYDVPDYAR_(n)YYY (SEQ ID NO: 69)YPYDVPDYAR_(n)LLL (SEQ ID NO: 64) MYPYDVPDYAR_(n)LLL (SEQ ID NO: 70)YPYDVPDYAR_(n)DDD (SEQ ID NO: 65) MYPYDVPDYAR_(n)DDD (SEQ ID NO: 71)YPYDVPDYAR_(n)KKK (SEQ ID NO: 66) MYPYDVPDYAR_(n)KKK (SEQ ID NO: 72)EDQVDPRLIDGKR_(n) (SEQ ID NO: 73) MEDQVDPRLIDGKR_(n) (SEQ ID NO: 79)EDQVDPRLIDGKR_(n)FFF (SEQ ID NO: 74) MEDQVDPRLIDGKR_(n)FFF (SEQ ID NO:80) EDQVDPRLIDGKR_(n)YYY (SEQ ID NO: 75) MEDQVDPRLIDGKR_(n)YYY (SEQ IDNO: 81) EDQVDPRLIDGKR_(n)LLL (SEQ ID NO: 76) MEDQVDPRLIDGKR_(n)LLL (SEQID NO: 82) EDQVDPRLIDGKR_(n)DDD (SEQ ID NO: 77) MEDQVDPRLIDGKR_(n)DDD(SEQ ID NO: 83) EDQVDPRLIDGKR_(n)KKK (SEQ ID NO: 78)MEDQVDPRLIDGKR_(n)KKK (SEQ ID NO: 84) QPELAPEDPEDR_(n) (SEQ ID NO: 85)MQPELAPEDPEDR_(n) (SEQ ID NO: 91) QPELAPEDPEDR_(n)FFF (SEQ ID NO: 86)MQPELAPEDPEDR_(n)FFF (SEQ ID NO: 92) QPELAPEDPEDR_(n)YYY (SEQ ID NO: 87)MQPELAPEDPEDR_(n)YYY (SEQ ID NO: 93) QPELAPEDPEDR_(n)LLL (SEQ ID NO: 88)MQPELAPEDPEDR_(n)LLL (SEQ ID NO: 94) QPELAPEDPEDR_(n)DDD (SEQ ID NO: 89)MQPELAPEDPEDR_(n)DDD (SEQ ID NO: 95) QPELAPEDPEDR_(n)KKK (SEQ ID NO: 90)MQPELAPEDPEDR_(n)KKK (SEQ ID NO: 96) HHHHHHR_(n) (SEQ ID NO: 97)MHHHHHHR_(n) (SEQ ID NO: 104) HHHHHHR_(n)FFF (SEQ ID NO: 98)MHHHHHHR_(n)FFF (SEQ ID NO: 105) HHHHHHR_(n)YYY (SEQ ID NO: 99)MHHHHHHR_(n)YYY (SEQ ID NO: 106) HHHHHHR_(n)LLL (SEQ ID NO: 101)MHHHHHHR_(n)LLL (SEQ ID NO: 107) HHHHHHR_(n)DDD (SEQ ID NO: 102)MHHHHHHR_(n)DDD (SEQ ID NO: 108) HHHHHHR_(n)KKK (SEQ ID NO: 103)MHHHHHHR_(n)KKK (SEQ ID NO: 109)

TABLE 3 Various Epitopes and Their Sequences Amino acid Name sequenceNucleotide sequence (5′→3′) His-6 HHHHHH CATCACCATCACCATCAC FLAGDYKDDDDK GACTACAAGGACGACGACGACAAG c-Myc EQKLISEEDLGAGCAGAAGCTGATCAGCGAGGAGGACC TG Strep- WSHPQFEK TGGAGCCACCCCCAGTTCGAGAAGTag Amber- CSPFEVQVSPEA TGCAGCCCCTTCGAGGTGCAGGTGAGCC 16 GAQKCCGAGGCCGGCGCCCAGAAG T7-Tag MASMTGGQQMG ATGGCCAGCATGACCGGCGGCCAGCAGATGGGC VSV-Gu^(#)- YTDIEMNRLGK TACACCGACATCGAGATGAACCGCCTGG Tag GCAAGHA*-Tag YPYDVPDYA TACCCCTACGACGTGCCCGACTACGCC Protein- EDQVDPRLIDGKGAGGACCAGGTGGACCCCCGCCTGATCG C Tag ACGGCAAG HSV^($)- QPELAPEDPEDCAGCCCGAGCTGGCCCCCGAGGACCCCG Tag AGGAC ^(#)Vesicular Stomatitis VirusGlycoprotein *HemAgglutinin ^($)Herpes Simplex Virus glycoprotein

1. A method comprising: a) providing: i) a first nucleic acid sequenceencoding a wild type polypeptide and a second nucleic acid sequenceencoding a truncated polypeptide of said wild type polypeptide, saidfirst and second sequences in a ratio greater than 250:1, said truncatedpolypeptide being between 10 and 150 amino acids in length; and ii) areconstituted in vitro translation system comprising purifiedrecombinant proteins; b) treating said reconstituted translation systemso as to reduce protease activity, wherein said treating comprises theaddition of one or more protease inhibitors which does not interferewith translation; c) introducing said nucleic acid sequences into saidreconstituted translation system under conditions such that saidtruncated polypeptide is produced; and d) determining the molecular massof said truncated polypeptide by mass spectrometry.
 2. The method ofclaim 1, wherein said nucleic acid is RNA.
 3. The method of claim 2,wherein said RNA is RNA made by in vitro transcription from a PCRproduct.
 4. The method of claim 3, wherein said PCR product is a PCRproduct amplified from genomic DNA obtained from a whole organism. 5.The method of claim 4, wherein said organism is a human subject.
 6. Themethod of claim 1, wherein said nucleic acid sequence comprises aportion complimentary to a portion of an APC gene.
 7. The method ofclaim 1, wherein said protease inhibitor is AEBSF.
 8. A methodcomprising: a) providing: i) a first nucleic acid sequence encoding awild type polypeptide and a second nucleic acid sequence encoding atruncated polypeptide of said wild type polypeptide, said truncatedpolypeptide being between 10 and 150 amino acids in length and beingassociated with cancer; and ii) a reconstituted in vitro translationsystem comprising purified recombinant proteins; b) treating saidreconstituted translation system so as to reduce protease activity,wherein said treating comprises the addition of one or more proteaseinhibitors which does not interfere with translation; c) introducingsaid nucleic acid sequences into said reconstituted translation systemunder conditions such that said truncated polypeptide is produced; andd) determining the molecular mass of said truncated polypeptide by massspectrometry.
 9. The method of claim 8, wherein said nucleic acid isRNA.